Antibodies and Uses Thereof

ABSTRACT

The present invention provides antibodies which bind to an epitope in the extracellular domain of human CC chemokine receptor 4 (CCR4) and which are capable of inhibiting the binding of macrophage-derived chemokine (MDC) and/or thymus and activation regulated chemokine (TARC) to CCR4. Also provided are inter alia immunoconjugates and compositions comprising such antibodies and methods and uses involving such antibodies, particularly in the medical and diagnostic fields.

This application is a divisional of U.S. Ser. No. 13/313,541, filed 7Dec. 2011, which is a non-provisional of U.S. Ser. No. 61/420,370, filed7 Dec. 2010. These previous applications are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates generally to the fields of antibodies,CCR4 biology and related therapies. More particularly, it providesantibodies that bind to CCR4. Such anti-CCR4 antibodies have diagnosticand therapeutic uses in diseases and conditions associated with CCR4,such as in imaging tumour blood vessels, treating cancer and treatingviral and other infections, and inflammatory and immune diseases. Theantibody-based compositions and methods of the invention also extend tothe use of immunoconjugates and other therapeutic combinations, kits andmethods.

BACKGROUND

With more than 800 members, G-protein-coupled receptors (GPCRs)represent the largest family of cell surface molecules involved insignal transmission, accounting for >2% of the total genes encoded byhuman genome. Members of the GPCR superfamily share a common membranetopology: an extracellular N-terminus, an intracellular C-terminus andseven transmembrane (TM) helices, which are connected by threeintracellular loops and three extracellular loops. On the basis of theirshared topological structure, GPCRs are also referred to as seventransmembrane (7TM) receptors. These receptors control key physiologicalfunctions, including neurotransmission, hormone and enzyme release fromendocrine and exocrine glands, immune responses, cardiac- andsmooth-muscle contraction and blood pressure regulation. Theirdysfunction contributes to some of the most prevalent human diseases.Emerging experimental and clinical data indicate that GPCRs have acrucial role in cancer progression and metastasis. Hence, there is thepossibility that some GPCRs may be suitable targets for anti-cancerdrugs.

Chemokines play an important role inter alia in immune and inflammatoryresponses in various diseases and disorders, including cancer, viralinfections, asthma and allergic diseases, as well as autoimmunepathologies such as rheumatoid arthritis and atherosclerosis. Thesesmall, secreted molecules are a growing superfamily of 8-14 kDa proteinscharacterised by a conserved four cysteine motif.

Studies have demonstrated that the actions of chemokines are mediated bysubfamilies of G protein-coupled receptors, among which is the receptordesignated chemokine (C—C motif) receptor 4, or CC chemokine receptor 4(CCR4). Specific ligands for CCR4 include the chemokines thymus andactivation-regulated chemokine (TARC) (also known as CCL17) andmacrophage-derived chemokine (MDC) (also known as CCL22). CCR4 may alsobind to RANTES, MCP-1 and MIP-1alpha, and CCR4 signalling in response tothese ligands has also been reported.

CCR4 is believed to be important inter alia in the function of T cellchemotaxis and the migration of phagocytic cells to sites ofinflammation. CCR4 is preferentially expressed on T-helper cell type 2(Th2) cells and regulatory T (Treg) cells, whereas only limitedexpression on other healthy cells or tissues occurs.

Tumour cells, in particular adult T cell leukaemia/lymphoma cells, maybe positive for CCR4. Expression of CCR4 by tumour cells is associatedwith skin involvement. Certain T-cell malignancies typically are locatedto the skin. For example, CCR4 is found at high levels in cutaneous Tcell lymphoma lesions.

More recently, CCR4 has also been found to be expressed by certain solidtumours (WO2009/037454). CCR4 expression is believed to be an earlyevent in carcinogenesis of solid tumours, particularly cancer of thecervix, oesophagus, kidney, brain, breast, ovary, prostate, stomach andpancreas. Thus, both haematological and non-haematological cancer cellsmay express CCR4. Consequently, these cancers maybe diagnosed, monitoredand treated using anti-CCR4 antibodies.

In addition, CCR4 has an important role in normal and tumour immunity. Asignificant fraction of CD4⁺CD25⁺ regulatory T-cells (Tregs) arepositive for CCR4 (Baatar et al, 2007b). These Tregs suppress immuneresponses through a variety of mechanisms, and it has been shown thatthey can inhibit tumour-specific immunity. Increased numbers of Tregsinfiltrating the stroma, the tumour itself, or draining lymphnodes,correlate with worsened outcome in a variety of cancers. Studies inmouse model show that reducing Treg activity leads to increasedendogenous anti-tumour immunity and increased efficacy of anti-tumourinterventions by the immune system. Consequently, inhibiting Tregfunction is a promising strategy in immunotherapy of tumours. Theinhibition can be achieved by killing the Tregs (depletion), interferingwith their suppressor functions, changing their trafficking pattern orchanging their differentiation.

In a subset of patients with CCR4+ T-cell leukaemia/lymphoma, the tumourcells themselves function as Treg cells, contributing to tumour survivalin the face of host antitumour immune responses. In other types ofcancers, MDC and TARC are produced by tumour cells and the tumourmicroenvironment and attract CCR4+ Treg cells to the tumour, where theycreate a favourable environment for tumour escape from the host immuneresponses. A higher frequency of Tregs in peripheral blood of patientswith following cancers has been reported: Breast cancer, Colorectalcancer, Oesophageal cancer, Gastric cancer, Hepatocellular carcinoma,Lung cancer, Melanoma, Ovarian cancer and Pancreatic cancer. Treg cellshave been reported to create a favourable environment for tumours.Hence, blocking the interaction between CCR4 and its ligands such as MDCcould be useful in the treatment or prevention of cancers, especiallythe cancers listed above. It has been reported that in a SCID mousemodel, antibody to human MDC/CCL22 was able to block infiltration ofhuman Treg cells into transplanted human ovarian tumours. It is believedthat the Treg cells present in human solid tumours prevent immuneeffector responses developing which could contribute to the slowing oftumour growth and metastasis. Thus, killing of Treg cells in the tumourmass, and/or prevention of migration of Treg cells to the tumour sitesby using a neutralising MAb (monoclonal antibody) directed against CCR4may result in enhanced immune responses towards solid tumours, and actas an adjunct to conventional cytotoxic or anti-hormonal therapies.

Cancer causes about 13% of all human deaths. According to the AmericanCancer Society, 7.6 million people died from cancer in the world during2007, so there remains a strong and urgent need for further anti-cancertherapeutics.

CCR4 has been shown to play a role in inflammation and immune disorders.Th2 cells and basophils are key cells in the allergic response in thelung and skin. There are a number of reports which describe the presenceof CCR4-expressing T cells and concomitant expression of CCR4 ligands(MDC, TARC) on airway epithelial cells in bronchial biopsies inallergen-challenged asthmatics (Panina-Bordignon et al, 2001). CCR4+ Tcells are also found in increased numbers in patients with atopicdermatitis, with a marked reduction of CCR4+ T cells observed when thedisease improved. Using a humanized SCID mouse model of asthma, it wasshown that blockade of CCR4 with antibodies prior to allergen challengereduced allergic airway inflammation, as well as the levels of Th2cytokines in the lungs. Depletion of CCR4+ T cells via lung delivery ofa blocking antibody may be a suitable treatment option for asthmaticpatients. Targeted delivery of a CCR4 blocking antibody to the skin mayalso be an attractive treatment for atopic dermatitis.

In allergic asthma, the presence of high levels of allergen-specific IgEis a reflection of an aberrant Th2 cell immune response to commonlyinhaled environmental allergens. Asthma is characterized by infiltrationof Th2 lymphocytes and eosinophils and by the production of Th2chemokines. Allergens are presented to T cells by dendritic cells (DCs)that continuously sample incoming foreign antigens. Upon properactivation by DCs, allergen-specific lymphocytes that are present indiseased airways produce Th2 cytokines interleukin (IL)-4, IL-5 andIL-13 that furthermore control leukocyte extravasation, goblet cellhyperplasia and bronchial hyper-reactivity (BHR). TARC and MDC producedby DCs induce the selective migration of Th2 cells but not Th1 cellsthrough triggering CCR4 (Perros et al, 2009). It was shown in murinemodels of asthma, that treatment with anti-TARC antibodies reduced thenumber of CD4+ T cells and eosinophils in bronchoalveolar lavage (BAL)fluid, the production of Th2 cytokines and airway hyper-responsivenessafter allergen challenge (Kawasaki et al, 2001). In contrast,CCR4-deficient mice showed no protection against airway inflammation andBHR (Chvatchko et al, 2000). Using a humanized SCID mouse model ofasthma, it was shown that blockade of CCR4 with antibodies prior toallergen challenge reduced allergic airway inflammation as well as thelevels of Th2 cytokines in the lungs (Perros et al, 2009). These dataindicate that CCR4 blockade is a feasible strategy for inhibitingallergic inflammation in humans.

Treg cells may suppress dendritic cells (DCs), thereby facilitating thedevelopment and progression of diseases, particularly infectiousdiseases and cancer. Anti-CCR4 antibodies able to block the suppressionof dendritic cells by Treg cells may therefore be useful as adjuvants invaccines, particularly as adjuvants in tumour vaccination or vaccinationagainst infectious disease. Thus, an anti-CCR4 antibody may enhance thetherapeutic effect of a vaccine, particularly enhancing thevaccine-induced immune response.

CCR4 binding compounds have been reported to show efficacy in murineallergic inflammation (Purandare et al, 2007, Burdi et al. 2007). It hasbeen reported that a CCR4-binding compound has reasonable potency invivo, as CCR4 dependent recruitment of leucocytes to the peritoneuminduced by TARC was inhibited by almost 90%. Yokoyama and colleaguespresented a quinazoline derivative targeting CCR4 which proved in vivoto be effective in reducing hypersensitivity reactions in a mouse model(Yokoyama et al, 2008b); a derivative of this compound proved to beeffective in a similar in vivo mouse model upon oral administration(Yokoyama et al, 2009). Recently, a group of scientists has identified anumber CCR4 antagonists using in silico modelling approach (Bayry et al,2008; Davies et al, 2009). By docking compounds to modelled CCR4, theauthors found molecules able to bind within the transmembrane region.Sixteen compounds inhibited CCR4-mediated migration of CCRF-CEM cells.When CCR4 antagonists were tested for their adjuvant function in vivowith Mycobacterium tuberculosis and hepatitis B vaccines, enhancedimmunogenicity was observed for both cellular and humoral immuneresponses. The observed effect was ascribed to inhibition of Tregactivity (Bayry et al, 2008; Davies et al, 2009). The fact that asignificant fraction of Treg cells are CCR4-positive is well known inthe art (Baatar et al 2007b). The observed effect is believed to beuseful not only in the context of vaccination against infectiousdiseases (caused, for example by a virus, a bacterium, a mycobacteriumor a parasite such as protozoa), but also in the context of cancervaccines.

As the cause for the efficacy of these compounds as adjuvants is basedon inhibiting Tregs by blocking CCR4 mediated signaling, it is expectedthat antibodies binding to CCR4 in an antagonistic manner would work thesame way; the pharmacological advantages of antibodies compared to smallmolecule drugs are well known in the art. The anti-CCR4 antibody KW-0761by Kyowa-Hakko is known in the art. However, this antibody is effectiveonly by ADCC; it does not prevent ligand-mediated signalling throughCCR4 receptor. Therefore, the antibodies described in this invention areexpected to be clearly superior in their modulation of immune reactionsvia Tregs.

Another application where modulating Tregs is of clinical use is cancertreatment. Tregs can inhibit tumour-specific immunity and theirincreased numbers correlate with unfavourable prognosis and diseaseprogression in some cancers. Studies in mouse models demonstrate thatreducing Treg activity boosts endogenous anti-tumour immunity, andincreases the efficacy of active immune interventions. Consequently,inhibiting Treg function is a strategy worth considering in human cancerimmunotherapy (Curiel, 2008; Ruter et al, 2009). This inhibition can beachieved both by modulating Tregs, or by directly killing them.

Examples for this approaches are described in the art by compoundstargeting other surface of Treg like CD25. Daclizumab (Zenapax®; Roche))and basiliximab (Simulect®; Novartis) are anti-human CD25 antibodiesapproved for use in autoimmune diseases, transplantation and cancersincluding HTLV-1 induced adult T-cell lymphoma/leukaemia (Church, 2003).Denileukin diftitox (Ontak®, DAB389IL-2; Ligand Pharmaceuticals Inc.) isa recombinant protein fusing the active domain of diphtheria toxin tohuman IL-2. In 1998, FDA has approved it to treat cutaneous T cellleukaemia/lymphoma (Olsen et al, 2001), which usually are CD4+CD25+.Denileukin diftitox is targeted to the IL-2 receptor and is proposed tobe internalized through CD25 by endocytosis. There is also evidence thatDenileukin diftitox improves immunogenicity of a tumour vaccine inpatients with renal cell cancer (Dannull et al, 2005). In addition, areport showed that denileukin diftitox reduces Treg numbers and functionin melanoma with improved melanoma-specific immunity (Mahnke et al,2007)

Other molecules on Tregs which are targeted for cancer treatment orimproved cancer vaccine effects include GITR (glucocorticoid-inducedtumour necrosis factor receptor-related gene) (Levings et al, 2002),Toll-like receptors (TLR) are expressed ubiquitously on a variety ofmammalian cells, including human Tregs (Yang et al, 2004, Rutter et al,2009) and Cytotoxic T lymphocyte antigen-4 (CTLA-4; CD152) (Sutmuller etal. 2001). Currently, Phase II and III clinical trials of anti-CTLA-4monoclonal antibody therapy are being conducted in melanoma, and Phase Iand II trials are being conducted in other tumour types. Two humanmonoclonal antibodies are under investigation—ipilimumab (MDX-010;Bristol-Myers Squibb/Medarex) and tremelimumab (CP-675,206; Pfizer).

CCR4 has also been implicated inter alia in the following disorders:Adult T-cell leukemia/lymphoma, Peripheral T-cell lymphoma, CutaneousT-Cell Lymphoma (CTCL), unspecified Diffuse large B-cell lymphoma,Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia, Epstein-Barrvirus (EBV) infection, Mycosis fungoides (a mature T-cell lymphoma),Sezary syndrome (a variant of mycosis fungoides), allergicbronchopulmonary aspergillosis (ABPA), Asthma, LPS-induced endotoxicshock, Allergic inflammation, T-cell mediated neurodegenerative diseasessuch as Multiple Sclerosis (MS), Autoimmune diseases such as Psoriasis,Castleman's disease and Rheumatoid arthritis (RA).

Due to their complex structures, GPCRs are considered as “difficulttargets” for raising specific antibodies. They can neither be easilypurified from the membrane fraction of lysed cells, nor be recombinantlyproduced in different expression systems as correctly folded solubleproteins. To the inventors' knowledge, to date all known attempts ofothers to generate anti-GPCR antibodies using phage display have provento be unsuccessful.

The difficulties associated with generating antibodies against GPCRs areset out in Hoogenboom et al., 1999. Furthermore, Sui et al. (2003)explain the difficulties associated with trying to obtain humanantibodies against the GPCR chemokine receptor CXCR4 and report thateven using the pathfinder method combined with step-back selection nospecific antibodies could be identified. Thus, in the field of GPCRs,the generation of specific antibodies remains a major challenge.

A murine monoclonal antibody called 1G1 which reacts with human CCR4 iscommercially available from BD Pharmingen. This antibody may be used forimmunoflurescent staining, but the antibody is not a neutralisingantibody.

A chimeric antibody to CCR4 designated KM2760 is disclosed in Ishida etal., 2006. The authors report that this antibody does not block thebinding of CCR4 to its ligands MDC or TARC.

The inventors have recognized that the identification of additionalantibodies that recognize CCR4 would be of benefit in expanding thenumber of therapeutic options. In particular, antibodies that block thebinding of CCR4 to one or more of its ligands would offer furthertherapeutic avenues.

The inventors have also recognized that the development of therapeuticagents for the treatment of humans that are better tolerated from animmunological perspective would be advantageous. In this regard, humanantibodies generally have at least three potential advantages for use inhuman therapy. First, the human immune system should not recognize theantibody as foreign. Second, the half-life in the human circulation willbe similar to naturally occurring human antibodies, allowing smaller andless frequent doses to be given. Third, because the effector portion ishuman, it will interact better with the other parts of the human immunesystem.

The art therefore still lacks anti-CCR4 antibodies that can be used inthe safe and effective treatment of patients having disorders in whichCCR4 is involved, including in long-term administration, and poseschallenges to the development of such antibodies.

In particular, there is a need for human antibodies to CCR4. Althoughhuman antibodies are generally recognized to display advantages, it isknown that the development of human antibodies that have high enoughaffinities and appropriate functional properties to make them candidatesfor successful human therapy is by no means straightforward. This iseven more so the case with GPCRs, due to their complex and transmembranenature.

There also remains a strong need for anti-CCR4 antibodies which canblock the binding between CCR4 and one or more of its ligands such asMDC and/or TARC.

DESCRIPTION OF THE INVENTION

The present invention overcomes certain limitations in the prior art byproviding new therapeutic compositions and methods for use in the safeand effective treatment of tumors, viral infections and other diseasesand conditions in which CCR4+ cells are involved such as inflammatory orimmune disorders. The invention is based on antibodies that bind toCCR4, preferably to an epitope within the extracellular domain of CCR4,particularly human antibodies. Such antibodies are effective in treatingtumors and viral infections and other diseases and conditions in whichCCR4+ cells are involved, such as inflammatory or immune disorders. Thecompositions and methods of the invention also extend to the use ofimmunoconjugates and combinations using this particular category ofantibodies.

A particular advantage of the present invention is that the antibodiesprovided inhibit binding of MDC and/or TARC to CCR4. This contrasts withthe leading antibodies in the clinical field, which do not inhibitbinding of MDC and/or TARC to CCR4.

The present inventors have prepared CCR4-specific antibodies that bindto CCR4. For example, the antibodies bind to CCR4+ cells, in particularHEK293T-cells transfected with CCR4, DT40-cells transfected with CCR4and CCRF-CEM cells which naturally express CCR4, as well as thefollowing cells which express CCR4: Hut78 (cutaneous T-cell lymphoma,ATCC number TIB-161), 786-0 (human renal cell carcinoma, ATCC numberCRL-1932), MCF-7 (human breast adenocarcinoma, ATCC number HTB-22),KatoIII (human gastric cancer, ATCC number HTB-103), L-428 cells(Non-Hodgkin Lymphoma, DSMZ number ACC 197) and A-498 (human renal cellcarcinoma, DSMZ number ACC 55)(see Example 2). Importantly, theantibodies do not significantly bind to CCR4− cells, i.e. cells which donot express CCR4. Thus, the antibodies disclosed herein bindspecifically to CCR4, making them suitable candidates for diagnosticsand therapy of the conditions discussed herein.

Amino acid and/or DNA sequences of preferred antibody molecules of theinvention which bind to an epitope in the extracellular domain of CCR4,their VH and VL domains including complementarity determining regions(CDRs), are set forth in the various SEQ ID NOs. listed herein.

Thus, the present invention provides an antibody which binds to CCR4 andwhich is capable of inhibiting the binding of MDC and/or TARC to CCR4.Preferably, the antibody is isolated. Also preferably, the antibody ishuman. Preferably, the antibody binds to an epitope in the extracellulardomain of CCR4. The CCR4 is preferably human. Thus, any reference to“binding to CCR4” includes the preferred embodiment of “binding to anepitope in the extracellular domain of CCR4”.

Thus, the invention preferably provides an isolated human antibody whichbinds to an epitope in the extracellular domain of human CCR4 and whichis capable of inhibiting the binding of MDC and/or TARC to CCR4.

In one embodiment, the present invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC and/orTARC to CCR4 comprising at least a heavy chain CDR comprising one of theheavy chain CDR sequences listed in the Tables disclosed herein, and/orat least a light chain CDR comprising one of the light chain CDRsequences listed in the Tables disclosed herein. Preferably, theantibody comprises at least 2, or 3 heavy and/or light chain CDRs, eachcomprising a relevant sequence listed in the Tables disclosed herein.

In one embodiment, the antibody of the invention comprises one or more,e.g. at least 2, 3, 4 or 5, preferably 6 of the CDRs selected from thegroup consisting of SEQ ID NOs: 1, 2, 3, 4, 5 and 6; or 1, 2, 3, 17, 5and 6; or 1, 2, 20, 22, 5 and 24; or 26, 27, 20, 22, 5, 24 or sequencessubstantially homologous to any one of the foregoing SEQ ID NOs.

In one embodiment, the antibody comprises a heavy chain CDR1 of SEQ IDNO: 1 or 26, a heavy chain CDR2 of SEQ ID NO: 2 or 27 and a heavy chainCDR3 of SEQ ID NO: 3 or 20, or sequences substantially homologous to anyone of the foregoing SEQ ID NOs, and/or a a light chain CDR1 of SEQ IDNO: 4, 17 or 22, a light chain CDR2 of SEQ ID: 5 and/or a light chainCDR3 of SEQ ID: 6 or 24, or sequences substantially homologous to anyone of the foregoing SEQ ID NOs.

Thus, in one embodiment the antibody comprises heavy chain CDR1 of SEQID NO: 26, a heavy chain CDR2 of SEQ ID NO: 2 and a heavy chain CDR3 ofSEQ ID NO: 20. Preferably, said antibody also comprises light chain CDRsselected from the sequences set out herein.

In one embodiment, the heavy chain CDR1 of any of the of the antibodiesdisclosed herein has the sequence SYAXS, wherein X may be any amino acid(SEQ ID NO: 119), preferably M or I (SEQ ID NO: 120); and/or the heavychain CDR2 of any of the of the antibodies disclosed herein has thesequence GIIPIFGTXNYAQKFQG, wherein X may be any amino acid (SEQ IDNO:121), preferably V, I or A (SEQ ID NO: 122); and/or the heavy chainCDR3 of any of the of the antibodies disclosed herein has the sequenceRX₁GX₂X₃FDY, wherein each X may independently be any amino acid (SEQ IDNO: 123), preferably X₁ is R or G, and/or X₂ is S or A, and/or X₃ is Yor K, most preferably X₁ is R or G, and X₂ is S or A, and X₃ is Y or K(SEQ ID NO: 124); and/or the light chain CDR1 of any of the of theantibodies disclosed herein has the sequence SGSTSNIGSHYVX, wherein X isany amino acid (SEQ ID NO: 125), preferably F, S or V (SEQ ID NO:126);and or the light chain CDR2 of any of the of the antibodies disclosedherein has the sequence of SEQ ID NO: 5; and/or the light chain CDR3 ofany of the of the antibodies disclosed herein has the sequence A V W D XX X X G W V, wherein each X may independently be any amino acid (SEQ IDNO:127), preferably X₁ is A or D, and/or X₂ is K or T, and/or X₃ is Y orL, and/or X₄ is R or S, more preferably X₁ is A or D, and X₂ is K or T,and X₃ is Y or L, and X₄ is R or S (SEQ ID NO:128).

This applies mutatis mutandis to the VH, VL, scFv and IgG sequencesdisclosed herein. Thus, any of the sequences disclosed herein whichinclude CDR sequences preferably include one or more of SEQ ID NOs119-128 instead of the corresponding CDR sequences listed in Tables 1-9.

In one especially preferred embodiment, the antibody comprises a heavychain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2 and aheavy chain CDR3 of SEQ ID NO: 3 or 20. Preferably, said antibody alsocomprises a light chain CDR1 of SEQ ID NO: 4, 17 or 22, a light chainCDR2 of SEQ ID: 5 and/or a light chain CDR3 of SEQ ID: 6 or 24, orsequences substantially homologous to any one of the foregoing SEQ IDNOs.

In one embodiment, the antibody comprises a heavy chain CDR1 of SEQ IDNO: 26, a heavy chain CDR2 of SEQ ID NO: 27 and a heavy chain CDR3 ofSEQ ID NO: 20. Preferably, said antibody also comprises a light chainCDR1 of SEQ ID NO: 4, 17 or 22, a light chain CDR2 of SEQ ID: 5 and/or alight chain CDR3 of SEQ ID: 6 or 24, or sequences substantiallyhomologous to any one of the foregoing SEQ ID NOs.

In one embodiment, the antibody comprises a light chain CDR1 of SEQ IDNO: 4, 17 or 22, a light chain CDR2 of SEQ ID: 5 and a light chain CDR3of SEQ ID: 6 or 24. Preferably, said antibody also comprises a heavychain CDR1 of SEQ ID NO: 1 or 26, a heavy chain CDR2 of SEQ ID NO: 2 or27 and/or a heavy chain CDR3 of SEQ ID NO: 3 or 20, or sequencessubstantially homologous to any one of the foregoing SEQ ID NOs.

In one embodiment, the antibody comprises a light chain CDR1 of SEQ IDNO: 4 or 17, a light chain CDR2 of SEQ ID: 5 and a light chain CDR3 ofSEQ ID: 6. Preferably, said antibody also comprises a heavy chain CDR1of SEQ ID NO: 1 or 26, a heavy chain CDR2 of SEQ ID NO: 2 or 27 and/or aheavy chain CDR3 of SEQ ID NO: 3 or 20, or sequences substantiallyhomologous to any one of the foregoing SEQ ID NOs.

In one embodiment, the antibody comprises a light chain CDR1 of SEQ IDNO: 22, a light chain CDR2 of SEQ ID: 5 and a light chain CDR3 of SEQID: 24. Preferably, said antibody also comprises a heavy chain CDR1 ofSEQ ID NO: 1 or 26, a heavy chain CDR2 of SEQ ID NO: 2 or 27 and/or aheavy chain CDR3 of SEQ ID NO: 3 or 20, or sequences substantiallyhomologous to any one of the foregoing SEQ ID NOs.

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO: 1, a heavy chain CDR2 domain of SEQ ID NO: 2, and aheavy chain CDR3 domain of SEQ ID NO: 3; and comprise

a light chain CDR1 domain of SEQ ID NO: 4, a light chain CDR2 domain ofSEQ ID NO: 5, anda light chain CDR 3 domain of SEQ ID NO: 6.

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO: 1, a heavy chain CDR2 domain of SEQ ID NO: 2, and aheavy chain CDR3 domain of SEQ ID NO: 3; and comprise

a light chain CDR1 domain of SEQ ID NO: 17, a light chain CDR2 domain ofSEQ ID NO: 5, anda light chain CDR 3 domain of SEQ ID NO: 6.

Certain most preferred antibodies comprise a heavy chain CDR1 domain ofSEQ ID NO: 1, a heavy chain CDR2 domain of SEQ ID NO: 2, and a heavychain CDR3 domain of SEQ ID NO: 20; and comprise

a light chain CDR1 domain of SEQ ID NO: 22, a light chain CDR2 domain ofSEQ ID NO: 5, anda light chain CDR 3 domain of SEQ ID NO: 24.

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO: 26, a heavy chain CDR2 domain of SEQ ID NO: 27, anda heavy chain CDR3 domain of SEQ ID NO: 20; and comprise

a light chain CDR1 domain of SEQ ID NO: 22, a light chain CDR2 domain ofSEQ ID NO: 5, anda light chain CDR 3 domain of SEQ ID NO: 24.

In a further embodiment, the invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC and/orTARC to CCR4 and that comprises at least one heavy chain variable regionthat comprises three CDRs and at least one light chain variable regionthat comprises three CDRs, wherein said heavy chain variable regioncomprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 1,(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2, and(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 3; and/or

said light chain variable region comprises:

(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 4, 17 or22, preferably 4 or 17(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 5, and(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 6 or 24,preferably 6.

In a further embodiment, the invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC to CCR4and that comprises at least one heavy chain variable region thatcomprises three CDRs and at least one light chain variable region thatcomprises three CDRs, wherein said heavy chain variable regioncomprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 1,(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2, and(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 20;and/orwherein light chain variable region comprises:(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 4, 17 or22, preferably 22,(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 5, and(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 6 or 24,preferably 24.

In a further embodiment, the invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC to CCR4and that comprises at least one heavy chain variable region thatcomprises three CDRs and at least one light chain variable region thatcomprises three CDRs, wherein said heavy chain variable regioncomprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 26,(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 27, and(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 20;and/or whereinsaid light chain variable region comprises:(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 4, 17 and22, preferably 22(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 5, and(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 6 or 24,preferably 24.Certain preferred embodiments of the invention provide an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDCand/or TARC to CCR4 comprising a VH domain that has the amino acidsequence of SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43 or 45 or asequence substantially homologous thereto and/or a VL domain that hasthe amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44 or46 or a sequence substantially homologous thereto.Preferably, said VH domain has 3 heavy chain CDRs and/or said VL domainhas 3 light chain CDRs. More preferably, at least 1, at least 2,preferably 3 of the light chain CDR sequences are selected from thelight chain CDR sequences disclosed herein and/or at least 1, at least2, preferably 3 of the heavy chain CDR sequences are selected from theheavy chain CDR sequences disclosed herein.

Thus, in one embodiment, said VH domain has a sequence of SEQ ID NO: 29,31, 33 or 35 or a sequence substantially homologous thereto andcomprises a heavy chain CDR1 domain of SEQ ID NO: 1, a heavy chain CDR2domain of SEQ ID NO: 2, and a heavy chain CDR3 domain of SEQ ID NO: 3.

In one embodiment said VH domain has a sequence of SEQ ID NO: 37, 39, 41or 43 or a sequence substantially homologous thereto and comprises aheavy chain CDR1 domain of SEQ ID NO: 1, a heavy chain CDR2 domain ofSEQ ID NO: 2, and a heavy chain CDR3 domain of SEQ ID NO: 20.

In one embodiment said VH domain has a sequence of SEQ ID NO: 45 or asequence substantially homologous thereto and comprises a heavy chainCDR1 domain of SEQ ID NO: 26, a heavy chain CDR2 domain of SEQ ID NO:27, and a heavy chain CDR3 domain of SEQ ID NO: 20.

In one embodiment said VL domain has a sequence of SEQ ID NO: 30 or 34or a sequence substantially homologous thereto and comprises a lightchain CDR1 domain of SEQ ID NO: 4, a light chain CDR2 domain of SEQ IDNO: 5, and a light chain CDR 3 domain of SEQ ID NO: 6.

In one embodiment said VL domain has a sequence of SEQ ID NO: 32 or 36or a sequence substantially homologous thereto and comprises a lightchain CDR1 domain of SEQ ID NO: 17, a light chain CDR2 domain of SEQ IDNO: 5, and a light chain CDR 3 domain of SEQ ID NO: 6.

In one embodiment said VL domain has a sequence of SEQ ID NO: 38, 40,42, 44 or 46 or a sequence substantially homologous thereto andcomprises a light chain CDR1 domain of SEQ ID NO: 22, a light chain CDR2domain of SEQ ID NO: 5, and a light chain CDR 3 domain of SEQ ID NO: 24.

The antibody may have any combination of the VL and VH sequencesdiscussed above. The following combinations are preferred:

A VH domain that has the amino acid sequence of SEQ ID NO: 29 or asequence substantially homologous thereto and a VL domain that has theamino acid sequence of SEQ ID NO: 30 or a sequence substantiallyhomologous thereto.A VH domain that has the amino acid sequence of SEQ ID NO: 31 or asequence substantially homologous thereto and a VL domain that has theamino acid sequence of SEQ ID NO: 32 or a sequence substantiallyhomologous thereto.A VH domain that has the amino acid sequence of SEQ ID NO: 33 or asequence substantially homologous thereto and a VL domain that has theamino acid sequence of SEQ ID NO: 34 or a sequence substantiallyhomologous thereto.A VH domain that has the amino acid sequence of SEQ ID NO: 35 or asequence substantially homologous thereto and a VL domain that has theamino acid sequence of SEQ ID NO: 36 or a sequence substantiallyhomologous thereto.A VH domain that has the amino acid sequence of SEQ ID NO: 37 or asequence substantially homologous thereto and a VL domain that has theamino acid sequence of SEQ ID NO: 38 or a sequence substantiallyhomologous thereto.A VH domain that has the amino acid sequence of SEQ ID NO: 39 or asequence substantially homologous thereto and a VL domain that has theamino acid sequence of SEQ ID NO: 40 or a sequence substantiallyhomologous thereto.A VH domain that has the amino acid sequence of SEQ ID NO: 41 or asequence substantially homologous thereto and a VL domain that has theamino acid sequence of SEQ ID NO: 42 or a sequence substantiallyhomologous thereto.A VH domain that has the amino acid sequence of SEQ ID NO: 43 or asequence substantially homologous thereto and a VL domain that has theamino acid sequence of SEQ ID NO: 44 or a sequence substantiallyhomologous thereto.A VH domain that has the amino acid sequence of SEQ ID NO: 45 or asequence substantially homologous thereto and a VL domain that has theamino acid sequence of SEQ ID NO: 46 or a sequence substantiallyhomologous thereto.

In a yet further embodiment, the present invention provides an antibodythat binds CCR4 and which is capable of inhibiting the binding of MDCand/or TARC to CCR4 comprising the amino acid sequence of SEQ ID NO: 47(said antibody also being referred to herein as 208 scFv), SEQ ID NO: 48(said antibody also being referred to herein as 306 scFv), SEQ ID NO: 49(said antibody also being referred to herein as 308 scFv), SEQ ID NO: 50(said antibody also being referred to herein as 406 scFv), SEQ ID NO: 51(said antibody also being referred to herein as 501 scFv), SEQ ID NO: 52(said antibody also being referred to herein as 503 scFv), SEQ ID NO: 53(said antibody also being referred to herein as 601 scFv), SEQ ID NO: 54(said antibody also being referred to herein as 603 scFv), or SEQ ID NO:55 (said antibody also being referred to herein as 803 scFv), orcomprising a fragment of any thereof that binds CCR4 and which iscapable of inhibiting the binding of MDC and/or TARC to CCR4, or asequence substantially homologous to any of the above sequence.

The invention is exemplified by monoclonal antibodies 208, 306, 308,406, 501, 503, 601, 603 and 803, single chain forms of which are shownin Tables 1, 2, 3, 4, 5, 6, 7, 8 and 9 (SEQ ID NOs: 47, 48, 49, 50, 51,21, 53, 54 and 55 respectively). Full length IgG forms of antibodies208, 306, 308, 406, 501, 503, 601, 603 and 803 have been made and theirsequences are shown in Tables 10-18 respectively. The CDR domains, VHand VL domains of the 208, 306, 308, 406, 501, 503, 601, 603 and 803antibodies are shown in Tables 1 to 9. Antibodies comprising these CDRdomains and/or VH and/or VL domains (or sequences substantiallyhomologous thereto) are preferred aspects of the invention.

A preferred embodiment of the invention is a scFv form of the 208antibody comprising or consisting of SEQ ID NO: 47, which is preferablyencoded by SEQ ID NO: 56. Another preferred embodiment of the inventionis a scFv form of the 306 antibody comprising or consisting of SEQ IDNO: 48, which is preferably encoded by SEQ ID NO: 57. Another preferredembodiment of the invention is a scFv form of the 308 antibodycomprising or consisting of SEQ ID NO: 49, which is preferably encodedby SEQ ID NO: 58. Another preferred embodiment of the invention is ascFv form of the 406 antibody comprising or consisting of SEQ ID NO: 50,which is preferably encoded by SEQ ID NO: 59. Another preferredembodiment of the invention is a scFv form of the 501 antibodycomprising or consisting of SEQ ID NO: 51, which is preferably encodedby SEQ ID NO: 60. Another preferred embodiment of the invention is ascFv form of the 503 antibody comprising or consisting of SEQ ID NO: 52,which is preferably encoded by SEQ ID NO: 61. Another preferredembodiment of the invention is a scFv form of the 601 antibodycomprising or consisting of SEQ ID NO: 53, which is preferably encodedby SEQ ID NO: 62. Another preferred embodiment of the invention is ascFv form of the 603 antibody comprising or consisting of SEQ ID NO: 54,which is preferably encoded by SEQ ID NO: 63. Another preferredembodiment of the invention is a scFv form of the 803 antibodycomprising or consisting of SEQ ID NO: 55, which is preferably encodedby SEQ ID NO: 64.

Other preferred embodiments are IgG forms of the 208, 306, 308, 406,501, 503, 601, 603 and 803 antibodies, preferably full length IgG forms.The IgG1 form of any of these antibodies is most preferred.

Thus, another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 67 (amino acid)and/or a light chain of SEQ ID NO: 68 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 65and/or a light chain encoded by SEQ ID NO: 66.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 71 (amino acid)and/or a light chain of SEQ ID NO: 72 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 69and/or a light chain encoded by SEQ ID NO: 70.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 75 (amino acid)and/or a light chain of SEQ ID NO: 76 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 73and/or a light chain encoded by SEQ ID NO: 74.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 79 (amino acid)and/or a light chain of SEQ ID NO: 80 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 77and/or a light chain encoded by SEQ ID NO: 78.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 83 (amino acid)and/or a light chain of SEQ ID NO: 84 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 81and/or a light chain encoded by SEQ ID NO: 82.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 87 (amino acid)and/or a light chain of SEQ ID NO: 88 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 85and/or a light chain encoded by SEQ ID NO: 86.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 91 (amino acid)and/or a light chain of SEQ ID NO: 92 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 89and/or a light chain encoded by SEQ ID NO: 90. substantially identicalheavy chains and two substantially identical light chains.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 95 (amino acid)and/or a light chain of SEQ ID NO: 96 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 93and/or a light chain encoded by SEQ ID NO: 94.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 99 (amino acid)and/or a light chain of SEQ ID NO: 100 (amino acid). Also preferred isan IgG antibody which comprises a heavy chain encoded by SEQ ID NO: 97and/or a light chain encoded by SEQ ID NO: 98.

It is of course understood that full IgG antibodies will comprise twosubstantially identical heavy chains and two substantially identicallight chains.

It is believed that the antibodies of the invention may bind to adifferent epitope to the known anti-CCR4 antibody family which comprisesKM2160, KM3060, KM2760 and KW-0761. Antibody KM2160 is a murine antibodywhich was raised against a peptide fragment of CCR4 and which recognisesan epitope existing in a region of positions 2-29 from the N-terminalamino acid of human CCR4 (EP1270595). KM2760 is a chimeric version ofthe antibody having the same binding characteristics (EP1270595).Antibody KM3060 is identical to KM2760, except that it is highlyfucosylated (Niwa et al. 2004, Cancer Research 64, 2127-2133). KW-0761is a humanised version of KM2760 (Ishida et al. Annals of Oncology 2008,vol 19, supplement 4, 513).

KM2760 has been reported not to block the interaction between CCR4 andTARC or MDC (Ishida et al. 2006, Cancer Research 66 (11), pp 5716-5722),which is consistent with the applicant's findings using the equivalentantibody KM3060var (corresponding to KM3060, but potentially having adifferent sugar profile as it was expressed in a different host). Bycontrast, the antibodies of the present invention were found to blockthe interaction between CCR4 and MDC and the interaction between CCR4and TARC (see Example 3). This strongly suggests that the antibodies ofthe invention bind to a different epitope than the prior art familywhich comprises KM2160, KM3060, KM2760 and KW-0761.

Moreover, antibodies 208, 306, 308, 406, 501, 503, 601, 603 and 803 werefound to compete with one another for binding to CCR4, indicating thatthey bind to the same, similar or at least overlapping epitopes. None ofthese antibodies competes with KW-0761 for binding to CCR4, indicatingthat KW-0761 binds to a different epitope (Example 4).

It is also believed that the antibodies of the invention may bind to adifferent epitope to the commercially available anti-CCR4 antibody 1G1.BD Pharmingen make it clear on the technical data sheet for thisantibody that this antibody is not a neutralising antibody. By contrast,the antibodies of the present invention are capable of blocking thebinding of MDC and/or TARC to CCR4. This strongly suggests that theantibodies of the invention bind to a different epitope than the 1G1antibody.

Certain examples of substantially homologous sequences are sequencesthat have at least 80%, 85%, 90% or 95% identity to the amino acidsequences disclosed. In certain embodiments, the antibodies of theinvention that bind to CCR4 and which are capable of inhibiting thebinding of MDC and/or TARC to CCR4 comprise at least one light chainvariable region that includes an amino acid sequence region of at leastabout 70%, 75%, 80% or 85%, more preferably at least about 90%, morepreferably at least about 95%, more preferably at least about 96%, 97%or 98% and most preferably at least 99% amino acid sequence identity tothe amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44 or46; and/or at least one heavy chain variable region that includes anamino acid sequence region of SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43or 45.

In certain embodiments, the antibodies of the invention that bind toCCR4 and which are capable of inhibiting the binding of MDC and/or TARCto CCR4 comprise at least one heavy chain variable region that includesan amino acid sequence region of at least about 70%, 75%, 80% or 85%,more preferably at least about 90%, more preferably at least about 95%,more preferably at least about 96%, 97% or 98% and most preferably atleast 99% amino acid sequence identity to the amino acid sequence of SEQID NO: 29, 31, 33, 35, 37, 39, 41, 43 or 45; and/or at least one lightchain variable region that includes an amino acid sequence region of SEQID NO: 30, 32, 34, 36, 38, 40, 42, 44 or 46.

Other preferred examples of substantially homologous sequences aresequences containing conservative amino acid substitutions of the aminoacid sequences disclosed. Preferably, the substantially homologoussequence contains no more than 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 altered amino acids across the whole VH domain and/or no morethan 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 altered amino acidsacross the whole VL domain. In some embodiments, any such altered aminoacids, if present, are only found in the framework regions.

In some embodiments, the substantially homologous sequence contains nomore than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 altered amino acids in one ormore or each of the FR regions disclosed. Thus preferably thesubstantially homologous sequence contains no more than 10, 9, 8, 7, 6,5, 4, 3, 2 or 1 altered amino acids in the heavy FR1, heavy FR2, heavyFR3, heavy FR4, light FR1, light FR2, light FR3, and/or light FR4disclosed herein.

A preferred substantially homologous sequence of any of the sequencesdisclosed herein that comprise a light chain FR1 is one in which theamino acid at position 1 of the light chain FR1 (serine) is substitutedwith another amino acid, preferably glutamine (Q) and/or the amino acidat position 2 (tyrosine) of the light chain FR1 is substituted withanother amino acid, preferably serine (S). Thus, preferred sequencesthat are substantially homologous to SEQ ID NOs: 12, 21 or 25 preferablyhave one or more preferably both of these substitutions, so they mostpreferably start with amino acids QS instead of SY. Any sequences whichcomprise SEQ ID NOs 12, 21 or 25 preferably also have one or both ofthese substitutions. Thus, preferred sequences that are substantiallyhomologous to SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44 or 46, 47-55,67, 71, 75, 79, 83, 87, 91, 95 or 99 have another amino acid, preferablyQ, instead of S at position 1 of the FR1 and/or another amino acid,preferably S, instead or Y at position 2 of FR1

Thus, a preferred homologe of SEQ ID NO: 12 is X₁X₂VLTQPPSASGTPGQSVTISC,wherein each X may be independently be any amino acid (SEQ ID NO: 129),preferably X₁ is Q, and/or X₂ is S (SEQ ID NO: 130). A preferredhomologe of SEQ ID NO: 21 is X₁X₂VLTQQPSASGTPGQSVTISC, wherein each Xmay be independently be any amino acid (SEQ ID NO: 131), preferably X,is Q, and/or X₂ is S (SEQ ID NO: 132). This applies mutatis mutandis tothe VL, scFv and IgG sequences disclosed herein. Thus, any of thesequences disclosed herein which include FR1 sequences preferablyinclude one or more of SEQ ID NOs 129-132 instead of the correspondingFR1 sequences listed in Tables 1-9.

Other preferred examples of substantially homologous sequences aresequences containing up to 1, 2, 3 or 4 preferably up to 1 or 2, alteredamino acids in one or more of the CDR regions disclosed.

In all such embodiments, such alterations might be conservative ornon-conservative amino acid substitutions, or a mixture thereof andpreferred alterations are conservative amino acid substitutions. Thus,in one embodiment all altered amino acids are conservativesubstitutions.

A “conservative amino acid substitution”, as used herein, is one inwhich the amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Homology may be assessed by any convenient method. However, fordetermining the degree of homology between sequences, computer programsthat make multiple alignments of sequences are useful, for instanceClustal W (Thompson et al., 1994). If desired, the Clustal W algorithmcan be used together with BLOSUM 62 scoring matrix (Henikoff andHenikoff, 1992) and a gap opening penalty of 10 and gap extensionpenalty of 0.1, so that the highest order match is obtained between twosequences wherein at least 50% of the total length of one of thesequences is involved in the alignment. Other methods that may be usedto align sequences are the alignment method of Needleman and Wunsch(1970), as revised by Smith and Waterman (1981) so that the highestorder match is obtained between the two sequences and the number ofidentical amino acids is determined between the two sequences. Othermethods to calculate the percentage identity between two amino acidsequences are generally art recognized and include, for example, thosedescribed by Carillo and Lipton (1988) and those described inComputational Molecular Biology, Lesk, e.d. Oxford University Press, NewYork, 1988, Biocomputing: Informatics and Genomics Projects.

Generally, computer programs will be employed for such calculations.Programs that compare and align pairs of sequences, like ALIGN (Myersand Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) andgapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereuxet al., 1984) are also useful for this purpose. Furthermore, the Daliserver at the European Bioinformatics institute offers structure-basedalignments of protein sequences (Holm, 1993; 1995; 1998).

By way of providing a reference point, sequences according to thepresent invention having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% homology, sequence identity etc. may be determined using the ALIGNprogram with default parameters (for instance available on Internet atthe GENESTREAM network server, IGH, Montpellier, France).

The term “substantially homologous” also includes modifications orchemical equivalents of the amino acid and nucleotide sequences of thepresent invention that perform substantially the same function as theproteins or nucleic acid molecules of the invention in substantially thesame way.

Any substantially homologous antibody should retain the ability tospecifically bind to CCR4, and preferably to the same epitope thereof asrecognized by the antibody in question, for example, the same epitope orantigen recognised by the CDR domains of the invention or the V_(H) andV_(L) domains of the invention as described herein. Binding to the sameepitope/antigen can be readily tested by methods well known anddescribed in the art, e.g. using eptiope mapping or binding assays, e.g.a competition assay, ELISA assay or BIAcore assay. Thus, a personskilled in the art will appreciate that binding assays can be used toidentify other antibodies and antibody fragments with the same bindingspecificities as the antibodies 208, 306, 308, 406, 501, 503, 601, 603and 803 respectively. As exemplified, below, a competition binding assaycan be used to identify such other antibodies. The method describedbelow is only one example of a suitable competition assay. The skilledperson will be aware of other suitable methods and variations.

Preferably, any substantially homologous antibody should retain thefunctional capabilities of the antibody, e.g. as defined elsewhereherein. Retention of functional properties can readily be tested bymethods well known and described in the art.

In some embodiments, the substantially homologous sequence lacks one ormore of the following sites compared with the sequences disclosedherein:

a sequence susceptible to a post-translational modification such asde-amidation and/or glycosylation;

a sequence susceptible to proteolytic digestion; and/or

a sequence capable of binding to human MHC classII receptors, i.e. a Tcell epitopes.

In silico testing may, for example, be carried out to determine thesusceptibility of the antibody sequences to post-translationalmodifications such as de-amidation, glycosylation, to proteolyticdigestion, and/or binding to human MHC classII receptors. Binding tohuman MHC classII receptors may cause T cell responses in humans, so itmay be useful to identify potential T cell epitopes. Amino acids orpeptide sequences identified as being putative sites susceptible topost-translational modifications, proteolytic digestion, or putative Tcell epitopes, may then be modified to alter their susceptibility. Thus,the substantially homologous sequences may contain one or more of suchmodifications.

In silico testing for T cell epitopes may, for example, involve aprogram termed iTOPE™ (Antitope, Cambridge, UK). iTope™ is a molecularmodelling technology which models the binding of peptides to 34 MHCclass II alleles. The contribution of individual amino acids to peptidebinding can be determined for each allele, and this provides informationon the precise location of the core 9 mer sequences that interact withthe MHC class II binding groove.

Alternatively or in addition, sequences may be analyzed using the TCED(T-cell epitope database) program (Antitope, Cambridge, UK) whichidentifies T-cell epitopes in antibody variable regions and which canalso be interrogated by BLAST searching to identify common motifs.

Epitope mapping can be performed using standard techniques. By way ofexample, the following methods for the identification and definition ofepitopes are mentioned herein. The amino acid sequence of CCR4 is known,so synthetic peptides may be used for epitope mapping, e.g. using thePepscan assay. Site directed mutagenesis is also a powerful tool inepitope mapping and can be used to evaluate the role of single aminoacids in immune complex formation. Protein footprinting relies on thefact that the epitope is protected from cleavage when bound as anantibody-antigen complex. Enzyme linked immunosorbent assay (ELISA) andhaemaglutination and slot-blotting may also be used in epitope mapping.Crystallisation of the antigen with the antibody may be used to map anon-linear epitope. Protocols for carrying out such methods are widelyavailable and the skilled person will be aware of suitable alternativemethods of epitope mapping.

Before a competition assay is performed using flow cytometry, somequantities of the tested antibody should be labeled, e.g. bybiotinylation. The functionality (retention of the cell-bindingproperties) of the biotinylated product and the minimal concentration ofthe biotinylated antibody of the invention (Ab1) that gives sub-maximalbinding against a fixed number of CCR4+ cells is determined. A total of10⁶ cells are harvested from exponentially growing cultures andincubated with various antibody concentrations for a suitable period oftime at a suitable temperature, e.g. 1 hr at 4° C. The cells are washedand incubated with a suitable detection antibody for a suitable periodof time at a suitable temperature, e.g. an additional hour at 4° C.After washing, the cells are analyzed by flow cytometry. For each testantibody, a saturation curve is generated from the data by plottingmedian fluorescence intensity (MFI) against the antibody concentration.

For the competition assay, CCR4+ cells may be prepared as above andtreated in duplicate with a mixture of fixed concentration of labeled(biotinylated) antibody (bio-Ab1) and increasing concentrations ofnon-labeled competitive antibody. The fixed concentration is the minimalconcentration of antibody that generates reasonable fluorescence signalagainst a fixed number of tumor cells as determined above. Ideally, thisfixed concentration in nM should be below the affinity of the treatedantibody at equilibrium (KO. In this case the described method can beused for estimation of affinities of competitive antibodies (Schodin andKranz, 1993, J Biol Chem 268:25755-7). The antibody mixture is incubatedwith target cells for a suitable period of time at a suitabletemperature, e.g. 1 hr at 4° C. The cells are washed and the cellbinding of biotinylated antibody is revealed by incubation withFITC-labeled streptavidin. After subtracting the background fluorescence(PBS-5% FCS) from the median fluorescence reading for each test sample(bio-Ab1+Ab2), the percent of inhibition is calculated for each Ab2concentration “c” according to the formula: %inhibition=(1−MFI^(bio-Ab1+Ab2“c”)/MFI^(bio-Ab1))×100 is calculated.

In all embodiments, the antibodies containing substantially homologoussequences retain the ability to bind CCR4 and the ability to inhibit thebinding of MDC and/or TARC to CCR4.

Other embodiments of the present invention provide binding proteins thatbind to CCR4 and have the ability to inhibit the binding of MDC and/orTARC to CCR4 and that comprise an antibody of the invention, a VH or VLdomain of the invention, or one or more of the CDRs of the invention. Ina preferred embodiment, such binding proteins are antibodies.

Preferred antibodies of the invention comprise at least one heavy chainvariable region that comprises three CDRs and at least one light chainvariable region that comprises three CDRs. Exemplary and preferredsequences for these CDRs are described herein.

As used herein, the succinct term “CCR4”, unless otherwise specificallystated or made clear from the scientific terminology, means CC chemokinereceptor 4 (also known as CD194).

CCR4 may be free CCR4, e.g. recombinant or purified CCR4, but preferablyit is present in a native form, e.g. on the surface of a cell.

The antibodies or binding proteins of the invention can also bind tofragments of CCR4, in particular fragments comprising or consisting ofthe extracellular domain, or can bind to entities comprising CCR4 orfragments of CCR4. Indeed, the epitope of the antibodies of theinvention is located in the extracellular domain of CCR4.

“CCR4” may also refer to any form of CCR4, particularly as CCR4 isconserved across mammalian species. The antibodies or antibody fragmentsof the invention may thus bind to human, monkey (e.g. cynomolgusmonkey), cow (bovine), mouse, rat, hamster, ferret, guinea pig and/orrabbit CCR4, for example. Preferably, the antibodies or antibodyfragments of the invention will bind at least to human CCR4. Thus,unless stated otherwise, any reference herein to “CCR4” may be read tomean “human CCR4”. In certain preferred embodiments, the antibodies orantibody fragments of the invention will bind at least to human andmonkey (e.g. cynomologus monkey) CCR4. In other preferred embodimentsthe antibodies or antibody fragments of the invention will bind at leastto human and mouse CCR4. In other preferred embodiments the antibodiesor antibody fragments of the invention will bind at least to human,monkey and mouse CCR4. In other preferred embodiments the antibodies orantibody fragments of the invention will bind at least to human, monkey,guinea pig and mouse CCR4. In some preferred embodiments, the antibodiesor antibody fragments of the invention will bind at least to human andmonkey CCR4, but not to murine CCR4. In other preferred embodiments, theantibodies or antibody fragments of the invention will bind to human andmonkey CCR4, but not to murine CCR4, e.g. only to human and monkey CCR4.

The Examples have shown that the antibodies of the invention can bind tohuman and monkey CCR4, and that they also have the ability to bind tomurine CCR4.

As used herein, the term “that binds to CCR4” or “anti-CCR4” in thecontext of antibodies or antibody fragments of the present invention,means antibodies or antibody fragments that are capable of one or moreof the following; preferably, of more than one of the following; andmost preferably, of all of the following:

(a) bind to CCR4 expressed on the surface of a cell, e.g. as assessed byflow cytometry or immunohistochemistry;(b) bind to a conformationally dependent (e.g. non linear) CCR4 epitope,e.g. as assessed by binding to CCR4 in a Western blot under non-reducingconditions;(c) bind to free CCR4; e.g. recombinantly expressed CCR4, on a solidsupport, e.g. as assessed by ELISA assay or BIAcore assay;(d) bind at least to human CCR4, more preferably to human and monkeyCCR4 or to human and mouse CCR4, most preferably to human, monkey andmouse CCR4 or to human and monkey CCR4 but not mouse CCR4;(e) bind to human CCR4 with a binding affinity (Kd) of 10 nM or less,preferably 5 nM or less, more preferably 3 nM or less or 2 nM or less,most preferably 1 nM or less as also discussed elsewhere herein;(f) bind to human and monkey CCR4 or to human and mouse CCR4, preferablyto human and monkey CCR4 but not mouse CCR4, with similar affinities,e.g. with a Kd of 10 nM or less, preferably 5 nM or less, morepreferably 3 nM or less or 2 nM or less, for example 1 nM or less asalso discussed elsewhere herein;(g) induce ADCC of CCR4+ cells as described elsewhere herein;(h) inhibit the binding of CCR4 to at least MDC and/or TARC, preferablyMDC and TARC, or preferably at least MDC and/or TARC and one or moreselected from RANTES, MCP-1 and MIP-1 alpha;(i) induce anti tumour effects in vivo;(j) localize to tumours upon administration to an animal with a tumour;(k) induce CDC of CCR4+ cells;(l) inhibit CCR4-mediated cellular responses to a CCR4 ligand,preferably inhibit the increase in intracellular calcium ionconcentration in response to a CCR4 ligand;(m) inhibit chemotaxis of CCR4+ cells towards a CCR4 ligand such as MDCand/or TARC.

In the context of binding to CCR4+ cells, it should be understood thatthe antibodies of the present invention bind to CCR4+ cells and do notsignificantly bind to CCR4⁻ cells (as shown in Example 2).

The term “do not significantly bind to CCR4⁻ cells” should be understoodsuch that any binding of the antibody to CCR4⁻ cells does not prohibitthe use of said antibody for therapeutic or diagnostic purposes. Thus,by “insignificant” binding to CCR4⁻ cells is meant that the binding ofthe antibody to CCR4⁻ cells is weaker than its binding to one or moreCCR4⁺ cells. Some cross-reaction with normal cells may thus occur, butthis level of binding can be considered to be “background” binding. Fortherapeutic or diagnostic purposes the main consideration is that theantibody must bind more strongly to one or more types of CCR4⁺ cellsthan to any CCR4⁻ with which the antibody may come into contact duringthe therapeutic or diagnostic application.

The antibody of the invention may be referred to as “CCR4-specific”. Theterm “CCR-specific” should be interpreted such that the binding of theantibody to CCR4 expressing cells is specific enough to allow the use ofsaid antibody for therapeutic or diagnostic purposes. The skilled personcan easily determine if any given antibody is CCR4-specific by comparingthe binding strength to the target CCR4⁺ cell with the binding strengthto one or more types of CCR4⁻ cells, e.g. wild-type (i.e. nottransformed with CCR4) HEK293T-cells or DT40-cells.

The skilled person will be aware that binding to CCR4⁺ cells compared toCCR4⁻ cells may be assessed, for example, using flow cytometry and asuitable example is described in Example 2.

Immunohistochemistry techniques, which are well known in the art, may beused to score the binding of antibodies to cells or samples. Such assaysmay be used to test the specificity of a particular antibody, or todetect CCR4 expression in tissue samples. Briefly, the antibody maytested for example on a high-density array of human tissues including apositive control (cells known to be CCR4-positive) and a negativecontrail (cells known to be CCR4-negative). The membranous stainingintensity may be estimated by visual inspection in a four step scale (0,1, 2, 3). Preferred antibodies show weak or strong, preferably strongimmunohistochemical scores for CCR4+ tissues.

The Examples show that binding of the anti-CCR4-antibodies to theCCR4-positive cell line CCRF-CEM is not inhibited in presence ofincreasing concentrations of human serum. Thus, the antibodies of theinvention can preferably bind to CCR4 in the presence of serum, theirbinding not being significantly inhibited by serum.

The binding profile of the anti-CCR4-antibodies of the invention to thedifferent CCR4+ cell lines tested in Example 2 shows increased bindingto some of the CCR4+ cell lines compared to other CCR4+ cell lines. Forinstance, binding to L-428 is decreased in comparison to CCRF-CEM.Without wishing to be bound by theory, this is believed to be becauseL-428 secrete the CCR4-ligand TARC and the anti-CCR4 antibodies of theinvention compete for the CCR4-binding site with the ligand (see Example3). Binding to CCR4 is therefore best assayed using cells that do notsecrete a CCR4 ligand.

The binding profile of the anti-CCR4-antibodies of the invention differsfrom that of KW0761. Without wishing to be bound by theory, this isbelieved to be due to different epitope binding sites of theseantibodies (as outlined in Example 4). KW0761 does not block the bindingof CCR4 and TARC, so it does not compete with the TARC secreted byL-428.

In addition, EC50 values were determined (data not shown) with varyingvalues from cell line to cell line, which is believed to be due todifferences in CCR4 expression on the surface of the various cell types.

Species cross-reactivity may be assayed using known methods, for exampleby flow cytometry using cells transfected with human CCR4 and CCR4 fromanother species respectively. A suitable assay is described in Example8, which shows that the antibodies of the invention can bind to human,monkey and murine CCR4.

The antibodies 208, 306, 308, 406, 501, 503, 601, 603 and 803 have beenshown to be capable of inhibiting the binding of CCR4 to its ligandsTARC and MDC (Example 3). Thus, preferably the antibodies of theinvention are capable of inhibiting the binding of CCR4 to one or moreof its ligands. Preferably, the binding to at least MDC is inhibited.More preferably, the binding to MDC and TARC is inhibited. In someembodiments, the binding of CCR4 to TARC is inhibited. In embodiments ofany of the aspects disclosed herein, the antibodies of the invention arecapable of inhibiting the binding of MDC and/or TARC to CCR4.

By the “inhibition of binding” of a ligand to CCR4 is meant that bindingof the ligand to CCR4 is reduced by at least 20, 30, or 40%, morepreferably at least 45, 50, 55, 60, 65, 70 or 75%, even more preferablyat least 80% in the presence of the antibody compared to binding in theabsence of the antibody. Embodiments in which the binding of ligand toCCR4 is reduced by at least 85, 90 or 95% are also contemplated.Alternatively viewed, when the ligand is first contacted with CCR4 andthe antibody is subsequently added, the ligand can inhibit the bindingof the antibody to CCR4.

Assays for determining whether an antibody can inhibit the binding of aligand to CCR4 are well known and a suitable assay is descried inExample 3. Briefly, CCR4+ cells were incubated with or withoutAlexa647-labelled MDC-SNAP, then antibody was added. Pre-incubation inthe presence of the MDC complex resulted in a reduction in antibodybinding to CCR4. Particularly, the binding of antibody to CCRF-CEM cells(which naturally express CCR4) and pre-incubated with MDC (or anappropriate MDC complex) is inhibited.

Alternative assays for determining whether an antibody can block thebinding of a ligand to CCR4 include the use of labeled ligand, e.g.radiolabelled ligand or biotinylated ligand and analysis with flowcytometry.

With regard to MDC, it should be noted that this agent is able to bindat least one GPCR receptor other than CCR4 with high affinity. Forexample, MDC has been reported to bind to the GPCR receptor D6 (Graham2009; Locati et al. 2005), and this may affect the outcome of bindingassays.

The antibodies of the invention are preferably capable of inhibitingCCR4-mediated cellular responses to a CCR4 ligand, in particular byinhibiting the increase in intracellular calcium ion concentration inresponse to a CCR4 ligand.

In particular, the antibodies are preferably capable of inhibitingMDC-induced calcium flux and/or TARC-induced calcium flux in CCRF-CEMcells (ATCC CCL-119). Suitable assay methods are known and an example ismentioned in Example 5.

Other preferred properties include the absence of significant toxicityin vivo when the antibodies of the invention are administered and theabsence of significant other side effects in vivo.

In some embodiments, the antibodies may inhibit chemotaxis of CCR4+cells towards a ligand of CCR4 such as MDC or TARC. This wasdemonstrated in Example 6.

In some embodiments, the antibodies may induce complement-dependentcytotoxicity (CDC) of CCR4+ cells, but in other embodiments theantibodies are not capable of inducing CDC. In some embodiments, theantibodies may induce apoptosis of CCR4+ cells, but in other embodimentsthe antibodies are not capable of inducing apoptosis. In someembodiments, the antibodies may be internalised by CCR4+ cells uponbinding to CCR4, but in other embodiments no significant internalisationtakes place.

The induction of apoptosis may be assayed using well-known standardmethods, for example methods which assay Annexin V staining. Briefly,cells may be incubated with an antibody for a suitable period of time,e.g. 24 hours and the effect, after cell harvesting and Annexin Vstaining may be measured by FACS analysis (e.g. using EasyCyte).

The induction of CDC may be assayed using well-known standard methods,for example methods which measure the relative number of viable cellsbased on the uptake and metabolism of a redox dye such as Alamar blue. Asuitable assay is disclosed in H Gazzano-Santoro et al. J ImmunolMethods. 1997, 28; 202(2):163-71.

The skilled person will be aware of suitable ways to assayinternalisation, for example using temperature-differential fluorescencelabeling on flow cytometry or confocal microscopy. An example of asuitable assay involves a secondary antibody labelled with apH-sensitive dye (such as CypHer5E), which is minimally fluorescent at abasic pH (as found outside of cells) and maximally fluorescent at anacidic pH (as found inside of cells).

The inhibition of chemotaxis may be assayed using standard methods, forexample using a transwell assay. Briefly, cells capable of chemotaxisand which express CCR4 are contacted with an antibody in one chamber anda ligand of CCR4 such as MDC is placed in another chamber separated fromthe first chamber by a membrane of filter having a suitable pore size.The effect of the antibody on cell migration towards the ligand(chemotaxis) is determined by comparing chemotaxis in the presence ofthe antibody to chemotaxis in the absence of the antibody. A suitablemethod is described in Example 6.

The term “ligand” of CCR4 includes the natural ligands of CCR4 such asMDC, TARC, RANTES, MCP-1 and/or MIP-1alpha, which may be naturallyproduced, recombinantly expressed or synthesised in the laboratory.

By “CCR4+ cells” is meant cells which express CCR4 on their surface,preferably at least substantially in its wild-type conformation. CCR4+cells may be naturally positive for CCR4, or they may be transformantswhich express recombinant CCR4. CCR4+ cells may inter alia include solidtumor cells, hematological tumor cells and/or Tregs. Preferred examplesof CCR4+ cells, particularly for in vitro assays, are CCRF-CEM, L-428,Hut78, 786-O, A498 and KatoIII.

In light of this invention, therefore, a range of anti-CCR4 antibodiescan be made and used in a variety of embodiments, including in thetreatment of any of the disorders discussed elsewhere herein,particularly cancer, immune disorders, inflammatory disorders andinfections.

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,except in instances wherein an upper limit is thereafter specificallystated. Therefore, an “antibody”, as used herein, means “at least afirst antibody”. The operable limits and parameters of combinations, aswith the amounts of any single agent, will be known to those of ordinaryskill in the art in light of the present disclosure.

Preferred embodiments of the invention are compositions comprising atleast one anti-CCR4 antibody of the invention, or antigen bindingfragment thereof.

Nucleic acid molecules comprising nucleotide sequences that encode theantibodies of the present invention as defined herein or parts orfragments thereof, or nucleic acid molecules substantially homologousthereto, form yet further aspects of the invention. The nucleic acidsequences disclosed in the Tables are preferred.

Preferred nucleic acid molecules comprise sequences which encode theamino acid sequence set out in SEQ ID NO: 47 (which is preferablyencoded by SEQ ID NO: 56 or a sequence substantially homologousthereto), SEQ ID NO: 48 (which is preferably encoded by SEQ ID NO: 57 ora sequence substantially homologous thereto), SEQ ID NO: 49 (which ispreferably encoded by SEQ ID NO: 58 or a sequence substantiallyhomologous thereto), SEQ ID NO: 50 (which is preferably encoded by SEQID NO: 59 or a sequence substantially homologous thereto), SEQ ID NO: 51(which is preferably encoded by SEQ ID NO: 60 or a sequencesubstantially homologous thereto), SEQ ID NO: 52 (which is preferablyencoded by SEQ ID NO: 61 or a sequence substantially homologousthereto), SEQ ID NO: 53 (which is preferably encoded by SEQ ID NO: 62 ora sequence substantially homologous thereto), SEQ ID NO: 54 (which ispreferably encoded by SEQ ID NO: 63 or a sequence substantiallyhomologous thereto) or SEQ ID NO: 55 (which is preferably encoded by SEQID NO: 64 or a sequence substantially homologous thereto).

Other preferred nucleic acid molecules comprise sequences which encode aheavy chain variable region (VH) that has the amino acid sequence of SEQID NO: 29, 31, 33, 35, 37, 39, 41, 43 or 45 or a sequence substantiallyhomologous thereto (which is preferably encoded by SEQ ID NO: 101, 103,105, 107, 109, 111, 113, 115 or 117 respectively or a sequencesubstantially homologous thereto) and/or comprise sequences which encodea light chain variable region (VL) which has the amino acid sequence ofSEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44 or 46 or a sequencesubstantially homologous thereto (which is preferably encoded by SEQ IDNO: 102, 104, 106, 108, 110, 112, 114, 116 or 118 respectively or asequence substantially homologous thereto).

More preferred are nucleic acids which encode the followingcombinations: SEQ ID NOs: 29 and 30; or SEQ ID NOs: 31 and 32; or SEQ IDNOs: 33 and 34; or SEQ ID NOs 35 and 36; or SEQ ID NOs 37 and 38; or SEQID NOs 39 and 40; or SEQ ID NOs 41 and 42; or SEQ ID NOs 43 and 44; orSEQ ID NOs 45 and 46. Also preferred are nucleic acid molecules whichcomprise the following combinations: SEQ ID NOs: 101 and 102; or SEQ IDNOs: 103 and 104; or SEQ ID NOs: 105 and 106; or SEQ ID NOs: 107 and108; or SEQ ID NOs: 109 and 110; or SEQ ID NOs: 111 and 112; or SEQ IDNOs: 113 and 114; or SEQ ID NOs: 115 and 116; or SEQ ID NOs: 117 and118.

As mentioned above, the FR1 region of the light chain may have residuesQS at positions 1 and 2. A preferred nucleic acid sequence is thereforeone which comprises or consists of a sequence which encodes a VLsubstantially homologous to VL 503 (SEQ ID NO: 40) having residues QS atpositions 1 and 2, preferably having the sequence set out below in whichthe changes compared to SEQ ID NO: 40 are underlined:

(SEQ ID NO: 133)           CAAAGCgtgctgactcagccaccctcagcgtctgggacccccgggcagagcgtcaccatctcttgttctggaagcacctccaacatcggaagtcattatgtggtctggtaccagcagctcccaggaacggcccccagactcctcatctataggaatcatcagcggccctcaggggtccctgaccgactctctggctccaagtctggcacctcagcctccctggccatcggtgggctccggtccgaggatgaggctgattattactgtgcagtgtgggatgacaccctgagtggctgggtgttcggcggagggaccaagctgaccgtccta.

Other preferred nucleic acid molecules comprise sequences that encodeIgG forms of the antibodies of the invention, for example those asdescribed in Example 1, or murine chimeric forms.

As indicated above, other nucleic acid molecules encompassed by thepresent invention are those encoding parts or fragments of the humanantibodies of the present invention, e.g., those encoding a heavy chainvariable region (VH) of an antibody or those encoding a light chainvariable region (VL) of an antibody. Other preferred nucleic acidmolecules are those encoding a heavy chain of an antibody of the presentinvention (e.g., those encoding SEQ ID NO: 67, 71, 75, 79, 83, 87, 91,95 or 99, such as SEQ ID NOs: 65, 69, 73, 77, 81, 85, 89, 93 or 97respectively or a sequence substantially homologous thereto) or thoseencoding a light chain of an antibody (e.g., those encoding SEQ ID NO:68, 72, 76, 80, 84, 88, 92, 96 or 100 such as SEQ ID NOs: 66, 70, 74,82, 86, 90, 94 or 98 respectively or a sequence substantially homologousthereto).

Thus, fragments of the antibodies of the invention as defined herein, orsequences substantially homologous thereto, or nucleic acid moleculescomprising sequences encoding such fragments form a yet further aspectof the invention.

The term “substantially homologous” as used herein in connection with anucleic acid sequence includes sequences having at least 70% or 75%,preferably at least 80%, and even more preferably at least 85%, 90%,95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid ornucleic acid sequence disclosed. Substantially homologous nucleic acidsequences of the invention thus include single or multiple basealterations (additions, substitutions, insertions or deletions) to thesequences of the invention.

Preferably, the antibodies of the present invention, when in IgG format,have a high binding affinity for CCR4, for example they may have a Kd inthe range of 1×10-8 M or 1×10-9 M or less. Importantly, antibodies withsuch an affinity are in the established range that has been shown to beuseful for therapy. Preferably, the antibodies of the invention, when inIgG format, have a binding affinity for CCR4 that corresponds to a Kd ofless than 30 nM, 20 nM, 15 nM or 10 nM, more preferably of less than 10,9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1nM, most preferably less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or0.1 nM.

Any appropriate method of determining Kd may be used. However,preferably the Kd is determined by testing various concentrations of thetest antibody against various concentrations of antigen (CCR4) in vitroto establish a saturation curve, for example using the Lineweaver-Burkmethod, or by using commercially available binding model software, suchas the 1:1 binding model in the BIAcore 1000 Evaluation software. Kdvalues may be calculated from IgG titrations on CCR+ cells using “onesite-specific binding” model f the software Prism (GraphPad, San Diego,Calif.).

With regard to determinations of Kd values, the skilled person willappreciate that apparent Kd values derived from binding experimentsusing cells expressing a target (e.g. CCR4) cannot be considered to bean absolute indication of affinity, because the experimental conditionswill affect the apparent binding affinity. For example, the levels ofexpression of CCR4 may vary depending on the conditions under which thecells are cultured, as well as differing between different cell types.It is consequently best to compare apparent Kd values obtained withinone set of experiments and it may not always be appropriate to compareKd values obtained in one set of experiments with Kd values obtained ina different set of experiments, particularly if the experimentalconditions varied significantly.

Alternatively, the off-rate and the antibody half-life on the surface ofthe CCR4-positive cell can be determined by performing the cell surfaceretention assay Adams et al., 1998, Prolonged in vivo tumour retentionof a human diabody targeting the extracellular domain of human HER2/neu.Br J Cancer 77: 1405-12; Le Gall et al., 1999, Di-, tri- and tetramericsingle chain Fv antibody fragments against human CD19: effect of valencyon cell binding. FEBS Lett 453: 164-8. The latter method allows moreappropriate mimicking the real situation in human patient under thetreatment conditions.

In some embodiments, antibodies of the invention may bind to both humanCCR4 and monkey CCR4. Such cross-reactivity between species and inparticular between humans and species commonly used as pre-clinicalanimal models may be an advantage as it allows a more effectivetranslation from pre-clinical studies to clinical use. For example,having an antibody which cross reacts with the native CCR4 present inthe particular animal model used means that the results in this modelare more likely to reflect the situation in a human patient, therebyallowing a more accurate assessment of for example dosing to be made andan increased likelihood of identifying any potentially relevant orproblematic side effects.

For example, the ability of an antibody of the invention to bind to bothhuman CCR4 and monkey CCR4 means that such antibodies can be tested inpreclinical toxicity studies to assess adverse side effects of thetreatment and to find appropriate tolerated dosages.

In addition, the ability to bind both human CCR4 and mouse CCR4 meansthat the results shown by such antibodies of the invention in mousemodels, e.g. mouse syngeneic models using immunocompetent mice, are morelikely to be representative of the activity of the antibodies in humansubjects. The reason for this is that antibodies which can bind to humanCCR4 but not mouse CCR4 will bind to CCR4 expressed by the human tumorcells in the mouse model but will not be able to bind to endogenousmurine CCR4. This is of course unlike the situation in a human patient,in which CCR4 expressed by the tumor and endogenous CCR4 would bepresent.

This is especially the case if the antibody has similar affinity to bothmurine and human CCR4.

The potential disadvantage with such a situation is that an antibodywhich binds to human CCR4 but not, or with significantly lower affinity,to mouse CCR4 might perform well in a human tumor xenograft model inimmunocompromized mice (e.g. nude or SCID mice) but this might not bereflected by a similar performance in a human system where much moreCCR4 was present. In other words, the anti-tumor effect seen in a mousexenograft system with an antibody which can bind to human CCR4 but notmouse CCR4 might look better than the clinical reality. In contrast,when working with an antibody that can bind to both human and mouse CCR4then this will bind to all forms of CCR4 present in the mouse modelsystem and is likely to be more representative of the situation when theantibody is put into humans. This is especially the case if the antibodyhas similar affinity to both murine and human CCR4.

In preferred embodiments, antibodies of the invention bind to human andmonkey CCR4 or to human and mouse CCR4 with similar affinities, e.g.with a Kd of 10 nM or less or 5 nM or less, more preferably 3 nM or lessor 2 nM or less, most preferably 1 nM or less.

By “similar affinity” is also meant that the binding affinity of theantibody for human CCR4 and for one or more of the other species ofinterest (e.g. monkey or mouse) is comparable, e.g. is not more than afactor of 20 different. More preferably the difference between thebinding affinities is less than a factor of 15, more preferably lessthan a factor of 10, most preferably less than a factor of 5, 4, 3 or 2.

However, in other embodiments the antibodies of the present inventionmay not bind to monkey CCR4 and/or they may not bind to mouse CCR4.

The antibodies of the invention bind to CCR4. Thus the antibodies orbinding proteins of the invention can be used to detect CCR4 in vivo orin vitro, in particular to detect CCR4+ cells. For example, as CCR4 isexpressed on certain tumour cells, the antibodies or binding proteins ofthe invention can be used to detect tumour cells in vivo or in vitro. Inaddition, the ability of the antibodies to localize to CCR4+ cells meansthat the antibodies of the invention can target body sites at whichCCR4+ cells are present, whereupon the antibody can act at the targetsite. In particular, the ability of the antibodies to localize to CCR4+tumour cells means that the antibodies of the invention can target bodysites at which CCR4+ tumour cells are present, whereupon the antibodycan act at the target site.

For example, the antibody may induce an anti-CCR4+ cell effect itselfi.e. as a naked antibody, e.g. by activating or inducing ADCC. Thisability to act as a naked Ab is advantageous. Alternatively, or inaddition, the antibody can induce an anti-CCR4+ cell effect by virtue ofbeing conjugated to an additional therapeutic molecule, e.g. a toxin orother anti-cancer molecule or an anti-inflammatory agent as describedherein.

The antibodies of the invention preferably have the ability to induceantibody dependent cellular cytotoxicity (ADCC) of CCR4+ cells. ADCC maybe assayed in vitro using methods well known in the art. Killing of theCCR4+ cell line CCRF-CEM in the presence of human PBMCs may be assayed,for example. A Chromium-51 release assay may be used, for example. Thus,the antibodies of the invention may for example cause at least 10%, 15%,20%, 22%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% killing of CCR4+cells in vitro, e.g. in the presence of human PBMCs. ADCC isadvantageous for some applications, particularly some therapeuticapplications. Thus, in preferred embodiments the antibody can induceADCC of CCR4+ cells, preferably of CCR4+ tumour cells and/or CCR4+ Th2cells. In some embodiments the antibody-mediated ADCC is in the presenceof PBMCs, but embodiments in which antibody-mediated ADCC is in theabsence of PBMCs are also contemplated. The ability of the antibodies ofthe invention to induce ADCC of CCR4+ cells was shown in Example 9. Theresults clearly demonstrate that the anti-CCR4 antibodies of theinvention are able to induce ADCC in the presence of human PBMCs on allthree target cell lines. The antibody 503 was determined to have an EC50of 5.3 pM when tested on CCRF-CEM cells, compared to 315 pM of KW0761.In addition, the anti-CCR4 antibodies of the invention also exhibitedcomparable maximum killing activities when challenged for ADCC onisolated Treg cells.

The antibodies of the invention are preferably also shown to be suitablypotent in terms of the concentration of antibody required to achievesuch ADCC levels. Thus, the antibody concentration required for halfmaximal cell lysis (EC50) of CCR4+ cells, e.g. CCRF-CEM cells, in vitrois preferably less than 700 ng/ml, 650 ng/ml, 620 ng/ml, 600 ng/ml, 550ng/ml, 500 ng/ml, 450 ng/ml, 400 ng/ml, 350 ng/ml, 300 ng/ml, 250 ng/ml,200 ng/ml, 150 ng/ml, 100 ng/ml, 90 ng/ml, 80 ng/ml, 70 ng/ml, 60 ng/ml,50 ng/ml, 46 ng/ml, 40 ng/ml, 35 ng/ml, 30 ng/ml, 25 ng/ml, 20 ng/ml, 15ng/ml, 10 ng/ml, 9 ng/ml, 7 ng/ml, 5 ng/ml, 2 ng/ml, 1 ng/ml, 0.5 ng/mlor 0.25 ng/ml.

Preferably, the above described abilities are observed at a measurableor significant level and more preferably at a statistically significantlevel, when compared to appropriate control levels.

It should be noted that PBMC (effector cells) prepared from differentdonors may exhibit a significant variation of ADCC with respect to theextent of non-specific and specific tumour cell lysis, as well as EC50values. This phenomenon has been described by Naundorf et al, 2002.

Human IgG1 is a glycoprotein bearing two N-linked oligosaccharide chainsbound to the Fc region. The oligosaccharides are of the complexbiantennary type, composed of a trimannosyl core structure with thepresence or absence of core fucose, bisecting N-acetylglucosamine(GlcNAc), galactose, and terminal sialic acid, which gives rise tostructural heterogeneity. Both human serum IgG and therapeuticantibodies are well known typically to be heavily fucosylated.

It has been reported that ADCC enhancement may in some instances beachieved by manipulating the state of oligosaccharides on human IgG1subclass. In particular, defucosylation has been shown to cause anincrease in ADCC activity of some antibodies (Niwa R et al, 2004). Thus,in preferred embodiments, antibodies according of the invention aremodified during production/expression of the protein, and/or in vitroafter production/expression, to generate a specific glycosylationpattern, particularly a glycosylation pattern which is beneficial fortherapeutic application of the antibodies. Preferably, said specificglycosylation pattern is the reduction or absence of fucose-basedglycosylation, which preferably increases the antibody's ability toinduce ADCC. Thus, in preferred embodiments, the antibodies of theinvention have a specific glycosylation pattern, preferably a specificglycosylation pattern which increases the ability of said antibody toinduce ADCC. Preferable, the antibodies of the invention aredefucosylated or non-fucosylated.

The skilled person is aware of suitable ways of preparing defucosylatedor non-fucosylated antibodies. For example, this can be achieved byproducing the antibody in presence of Kifunensine (for example 100ng/ml), a selective inhibitor of class I α-mannosidases, leading to adecrease in fucosylation of the molecule during production. Suitablehost cells which lack one or more proteins required for fucosylation ofoligosaccharide moieties can be used to produce defucosulatedantibodies, e.g. fucosyltransferase-deficient host cells. Examples ofsuitable host cells are cells wherein the activity of an enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose and/orthe activity of an enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through an α-bond in the complexN-glycoside-linked sugar chain is decreased or deleted. Examples of suchenzymes include enzymes relating to the synthesis of GDP-fucose includeGMD (GDP-mannose 4,6-dehydratase), Fx (GDP-keto-6-deoxymannose3,5-epimerase, 4-reductase), GFPP (GDP-beta-L-fucose pyrophosphorylase).

As described in Example 2, defucosylated version of antibodies 306, 406and 503 were produced in the presence of Kifunensine, a selectiveinhibitor of class I α-mannosidases, causing a stop in fucosylation ofthe IgG during production in cell culture.

By “defucosulated” is meant that at least 10%, preferably at least 20,30, 40 50, 60, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or atleast 99% of the total complex N-glycoside-linked sugar chains bound tothe Fc region are sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain. By“non-fucosylated” is meant that no significant levels of fucose arepresent in the antibody.

Some antibodies are capable of being internalized into the cells towhich they become bound. Thus, in some embodiments of the invention theantibodies are capable of being internalized. This property isparticularly advantageous for use in immunoconjugates as any other agentattached to the antibody molecule should be internalized with theantibody molecule. In other embodiments no significant internalizationis seen.

CCR4 is known to be expressed on platelets, and its ligands MDC and TARCare known to induce platelet aggregation. This could potentially beproblematic, as IgG molecules have two ligand binding sites, so there isa possibility that an IgG capable of recognizing CCR4 may be able tocross link platelets if both arms are able to bind to CCR4 on differentplatelets. This might result in blot clotting in vivo. Particularly formedical applications, it is desirable that the antibody does not induceany significant platelet aggregation. Platelet aggregation may beassayed using known methods. The effect of the antibodies of theinvention on platelet aggregation was assayed as described in Example11. Essentially, the antibodies were incubated with isolated plateletsalone or in combination ADP, a well-described inducer of aggregation(Varon and Spectre “Antiplatelet agents” Hematology Am Soc Hematol EducProgram. 267-72, 2009). Binding of the anti-CCR4 antibodies to plateletswas observed, but the antibodies were shown to have no effect onplatelet aggregation. They do not induce aggregation, nor do theyinhibit e.g. ADP-induced platelet aggregation. Thus, the antibodies donot have any significant effect on platelet aggregation.

As discussed above, certain PBLs, including Tregs, express CCR4, so insome embodiments of the invention the antibodies can bind to PBLs,preferably to Tregs and/or Th2 cells. This feature is advantageous,particularly in immunotherapy, as it may allow the depletion of Tregcells.

In the following descriptions of the compositions, immunoconjugates,pharmaceuticals, combinations, cocktails, kits, first and second medicaluses and all methods in accordance with this invention, the terms“antibody” and “immunoconjugate”, or an antigen-binding region orfragment thereof, unless otherwise specifically stated or made clearfrom the scientific terminology, refer to a range of anti-CCR4antibodies as well as to the specific 208, 306, 308, 406, 501, 503, 601,603 and 803 antibodies.

The terms “antibody” and “immunoglobulin”, as used herein, refer broadlyto any immunological binding agent or molecule that comprises a humanantigen binding domain, including polyclonal and monoclonal antibodies.Depending on the type of constant domain in the heavy chains, wholeantibodies are assigned to one of five major classes: IgA, IgD, IgE,IgG, and IgM and the antibodies of the invention may be in any one ofthese classes. Several of these are further divided into subclasses orisotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. The heavy-chainconstant domains that correspond to the difference classes ofimmunoglobulins are termed α, δ, ε, γ and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

Generally, where whole antibodies rather than antigen binding regionsare used in the invention, IgG and/or IgM are preferred because they arethe most common antibodies in the physiological situation and becausethey are most easily made in a laboratory setting. IgG1 antibodies areparticularly preferred.

The “light chains” of mammalian antibodies are assigned to one of twoclearly distinct types: kappa (κ) and lambda (λ), based on the aminoacid sequences of their constant domains and some amino acids in theframework regions of their variable domains. There is essentially nopreference to the use of κ or λ light chain constant regions in theantibodies of the present invention.

As will be understood by those in the art, the immunological bindingreagents encompassed by the term “antibody” extend to all antibodies andantigen binding fragments thereof, including whole antibodies, dimeric,trimeric and multimeric antibodies; bispecific antibodies; chimericantibodies; recombinant and engineered antibodies, and fragmentsthereof.

The term “antibody” is thus used to refer to any antibody-like moleculethat has an antigen binding region, and this term includes antibodyfragments that comprise an antigen binding domain such as Fab′, Fab,F(ab′)2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv(single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies,diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fabfusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); Bispecific T-cell Engager (BiTE)(scFv-scFv tandems to attract T cells); dual variable domain (DVD)-Ig(bispecific format); small immunoprotein (SIP) (kind of minibody); SMIP(“small modular immunopharmaceutical” scFv-Fc dimer; DART (ds-stabilizeddiabody “Dual Affinity ReTargeting”); small antibody mimetics comprisingone or more CDRs and the like.

The techniques for preparing and using various antibody-based constructsand fragments are well known in the art (see Kabat et al., 1991,specifically incorporated herein by reference). Diabodies, inparticular, are further described in EP 404, 097 and WO 93/11161;whereas linear antibodies are further described in Zapata et al. (1995).

Antibodies can be fragmented using conventional techniques. For example,F(ab′)2 fragments can be generated by treating the antibody with pepsin.The resulting F(ab′)2 fragment can be treated to reduce disulfidebridges to produce Fab′ fragments. Papain digestion can lead to theformation of Fab fragments. Fab, Fab′ and F(ab′)2, scFv, Fv, dsFv, Fd,dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques or can be chemically synthesized. Techniques forproducing antibody fragments are well known and described in the art.For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; LeGall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young etal., 1995 further describe and enable the production of effectiveantibody fragments.

The antibodies or antibody fragments can be produced naturally or can bewholly or partially synthetically produced. Thus the antibody may befrom any appropriate source, for example recombinant sources and/orproduced in transgenic animals or transgenic plants, or in eggs usingthe IgY technology. Thus, the antibody molecules can be produced invitro or in vivo.

Preferably, the antibody or antibody fragment comprises an antibodylight chain variable region (VL) that comprises three CDR domains and anantibody heavy chain variable region (VH) that comprises three CDRdomains. Said VL and VH generally form the antigen binding site.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region has a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions (CDRs) of each variable domain interact to definean antigen-binding site on the surface of the VH-VL dimer. Collectively,the six hypervariable regions (CDRs) confer antigen-binding specificityto the antibody.

However, it is well documented in the art that the presence of threeCDRs from the light chain variable domain and three CDRs from the heavychain variable domain of an antibody is not necessary for antigenbinding. Thus, constructs smaller than the above classical antibodyfragment are known to be effective.

For example, camelid antibodies (Hamers-Casterman et al., 1993; ArbabiGhahroudi et al., 1997) have an extensive antigen binding repertoire butare devoid of light chains. Also, results with single domain antibodiescomprising VH domains alone (Ward et al., 1989; Davies and Riechmann,1995) or VL domains alone (van den Beucken et al., 2001) show that thesedomains can bind to antigen with acceptably high affinities. Thus, threeCDRs can effectively bind antigen.

It is also known that a single CDR, or two CDRs, can effectively bindantigen. As a first example, a single CDR can be inserted into aheterologous protein and confer antigen binding ability on theheterologous protein, as exemplified by showing that a VH CDR3 regioninserted into a heterologous protein, such as GFP, confers antigenbinding ability on the heterologous protein (Kiss et al., 2006; Nicaiseet al., 2004).

It is further known that two CDRs can effectively bind antigen, and evenconfer superior properties than possessed by the parent antibody. Forexample, it has been shown (Qiu et al., 2007) that two CDRs from aparent antibody (a VH CDR1 and a VL CDR3 region) retain the antigenrecognition properties of the parent molecule but have a superiorcapacity to penetrate tumours. Joining these CDR domains with anappropriate linker sequence (e.g., from VH FR2) to orientate the CDRs ina manner resembling the native parent antibody produced even betterantigen recognition. Therefore, it is known in the art that it ispossible to construct antigen binding antibody mimetics comprising twoCDR domains (preferably one from a VH domain and one from a VL domain,more preferably, with one of the two CDR domains being a CDR3 domain)orientated by means of an appropriate framework region to maintain theconformation found in the parent antibody.

Thus, although preferred antibodies of the invention might comprise sixCDR regions (three from a light chain and three from a heavy chain),antibodies with fewer than six CDR regions and as few as one or two CDRregions are encompassed by the invention. In addition, antibodies withCDRs from only the heavy chain or light chain are also contemplated.

Preferred antibodies of the invention that bind to CCR4 comprise atleast one heavy chain variable region that comprises three CDRs and atleast one light chain variable region that comprises three CDRs, whereinsaid heavy chain variable region comprises heavy chain CDRs having thesequences disclosed herein.

Preferred light chain CDR regions for use in conjunction with thespecified heavy chain CDR regions are described elsewhere herein.However, other light chain variable regions that comprise three CDRs foruse in conjunction with the heavy chain variable regions of theinvention are also contemplated. Appropriate light chain variableregions which can be used in combination with the heavy chain variableregions of the invention and which give rise to an antibody which bindsCCR4 can be readily identified by a person skilled in the art.

For example, a heavy chain variable region of the invention can becombined with a single light chain variable region or a repertoire oflight chain variable regions and the resulting antibodies tested forbinding to CCR4. It would be expected that a reasonable number of suchcombinations of heavy chain variable regions of the invention withdifferent light chain variable regions would retain the ability to bindCCR4.

Similar methods could be used to identify alternative heavy chainvariable regions for use in combination with preferred light chainvariable regions of the invention.

In certain embodiments, the antibody or antibody fragment comprises allor a portion of a heavy chain constant region, such as an IgG1, IgG2,IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Preferably, theheavy chain constant region is an IgG1 heavy chain constant region, or aportion thereof. Furthermore, the antibody or antibody fragment cancomprise all or a portion of a kappa light chain constant region or alambda light chain constant region, or a portion thereof. All or part ofsuch constant regions may be produced naturally or may be wholly orpartially synthetic. Appropriate sequences for such constant regions arewell known and documented in the art. When a full complement of constantregions from the heavy and light chains are included in the antibodiesof the invention, such antibodies are typically referred to herein as“full length” antibodies or “whole” antibodies.

Antibodies containing an Fc region are preferred for certain uses,particularly therapeutic uses in vivo, where the Fc region mediateseffector functions such as ADCC.

The substantially homologous nucleic acid sequences also includenucleotide sequences that hybridize to the nucleic acid sequencesdisclosed (or their complementary sequences), e.g., hybridize tonucleotide sequences encoding one or more of the light chain or heavychain CDRs of the invention, the light or heavy chain variable regionsof the invention, or the antibodies of the invention (or hybridize totheir complementary sequences), under at least moderately stringenthybridization conditions, preferably highly stringent conditions.

Substantially homologous sequences of proteins of the invention alsoinclude, without limitation, alterations that do not affect the VH, VLor CDR domains of the antibodies, e.g., include scFv antibodies where adifferent linker sequence is used or antibodies where tag sequences orother components are added that do not contribute to the binding ofantigen, or alterations to convert one type or format of antibodymolecule or fragment to another type or format of antibody molecule orfragment (e.g., conversion from Fab to scFv or vice versa), or theconversion of an antibody molecule to a particular class or subclass ofantibody molecule (e.g., the conversion of an antibody molecule to IgGor a subclass thereof, e.g., IgG1 or IgG3).

In other preferred embodiments, second generation antibodies areprovided that have enhanced or superior properties in comparison to anoriginal anti-CCR4 antibody, such as 208, 306, 308, 406, 501, 503, 601,603 and 803. For example, the second generation antibodies may have astronger binding affinity for CCR4, a superior cross reactivity profile,superior ability to target CCR4+ cells, particularly tumour cells, animproved ability to induce ADCC, an improved ability to induce CDC, animproved treatment of the disorders discussed elsewhere herein.

Comparisons to identify effective second generation antibodies arereadily conducted and quantified, e.g., using one or more of the variousassays described in detail herein or in the art. Second generationantibodies that have an enhanced biological property or activity of atleast about 2-fold, 5-fold, 10-fold, 20-fold, and preferably, at leastabout 50-fold, in comparison to the anti-CCR4 antibodies of the presentinvention, as exemplified by the 208, 306, 308, 406, 501, 503, 601, 603and 803 antibody, are encompassed by the present invention.

The antibody, binding protein and nucleic acid molecules of theinvention are generally “isolated” or “purified” molecules insofar asthey are distinguished from any such components that may be present insitu within a human or animal body or a tissue sample derived from ahuman or animal body. The sequences may, however, correspond to or besubstantially homologous to sequences as found in a human or animalbody. Thus, the term “isolated” or “purified” as used herein inreference to nucleic acid molecules or sequences and proteins orpolypeptides, e.g., antibodies, refers to such molecules when isolatedfrom, purified from, or substantially free of their natural environment,e.g., isolated from or purified from the human or animal body (if indeedthey occur naturally), or refers to such molecules when produced by atechnical process, i.e., includes recombinant and synthetically producedmolecules.

Thus, when used in connection with a nucleic acid molecule, such termsmay refer to a nucleic acid substantially free of material with which itis naturally associated such as other nucleic acids/genes orpolypeptides. These terms may also refer to a nucleic acid substantiallyfree of cellular material or culture medium when produced by recombinantDNA techniques, or substantially free of chemical precursors, or otherchemicals when chemically synthesized. An isolated or purified nucleicacid may also be substantially free of sequences that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) from which the nucleic acid is derived or sequences thathave been made to flank the nucleic acid (e.g., tag sequences or othersequence that have no therapeutic value) by, for example, geneticengineering.

Thus, when used in connection with a protein or polypeptide moleculesuch as light chain CDRs 1, 2 and 3, heavy chain CDRs 1, 2 and 3, lightchain variable regions, heavy chain variable regions, and bindingproteins or antibodies of the invention, including full lengthantibodies, the term “isolated” or “purified” typically refers to aprotein substantially free of cellular material or other proteins fromthe source from which it is derived. In some embodiments, particularlywhere the protein is to be administered to humans or animals, suchisolated or purified proteins are substantially free of culture mediumwhen produced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. Such isolated or purifiedproteins may also be free of flanking sequences such as those describedabove for the isolated nucleic acid molecules.

The term “nucleic acid sequence” or “nucleic acid molecule” as usedherein refers to a sequence of nucleoside or nucleotide monomerscomposed of naturally occurring bases, sugars and intersugar (backbone)linkages. The term also includes modified or substituted sequencescomprising non-naturally occurring monomers or portions thereof. Thenucleic acid sequences of the present invention may be deoxyribonucleicacid sequences (DNA) or ribonucleic acid sequences (RNA) and may includenaturally occurring bases including adenine, guanine, cytosine,thymidine and uracil. The sequences may also contain modified bases.Examples of such modified bases include aza and deaza adenine, guanine,cytosine, thymidine and uracil; and xanthine and hypoxanthine. Thenucleic acid molecules may be double stranded or single stranded. Thenucleic acid molecules may be wholly or partially synthetic orrecombinant.

In preferred embodiments the antibodies of the invention are humanantibodies, more preferably fully human antibodies. In this regard,human antibodies generally have at least three potential advantages foruse in human therapy. First, the human immune system should notrecognize the antibody as foreign. Second, the half-life in the humancirculation will be similar to naturally occurring human antibodies,allowing smaller and less frequent doses to be given. Third, because theeffector portion is human, it will interact better with the other partsof the human immune system, e.g., to destroy target cells moreefficiently by complement-dependent cytotoxicity (CDC) orantibody-dependent cellular cytotoxicity (ADCC).

However, although human antibodies are generally recognized to displaythese advantages, it is known that the development of human antibodiesthat have high enough affinities and appropriate functional propertiesto make them candidates for successful human therapy is by no meansstraightforward. The art therefore still lacks anti-CCR4 for the safeand effective treatment of humans, and poses challenges to thedevelopment of such agents.

The term “human” as used herein in connection with antibody moleculesand binding proteins first refers to antibodies and binding proteinshaving variable regions (e.g., VH, VL, CDR or FR regions) and,optionally, constant antibody regions, isolated or derived from a humanrepertoire or derived from or corresponding to sequences found inhumans, e.g., in the human germline or somatic cells.

The 208, 306, 308, 406, 501, 503, 601, 603 and 803 antibodies areexamples of such a human antibody molecules wherein the variable regionshave been isolated from a human repertoire.

The “human” antibodies and binding proteins of the invention furtherinclude amino acid residues not encoded by human sequences, e.g.,mutations introduced by random or site directed mutations in vitro, forexample mutations introduced by in vitro cloning or PCR. Particularexamples of such mutations are mutations that involve conservativesubstitutions or other mutations in a small number of residues of theantibody or binding protein, e.g., in up to 5, 4, 3, 2 or 1 of theresidues of the antibody or binding protein, preferably e.g., in up to5, 4, 3, 2 or 1 of the residues making up one or more of the CDRs of theantibody or binding protein. Certain examples of such “human” antibodiesinclude antibodies and variable regions that have been subjected tostandard modification techniques to reduce the amount of potentiallyimmunogenic sites.

Thus, the “human” antibodies of the invention include sequences derivedfrom and related to sequences found in humans, but which may notnaturally exist within the human antibody germline repertoire in vivo.In addition, the human antibodies and binding proteins of the presentinvention include proteins comprising human consensus sequencesidentified from human sequences, or sequences substantially homologousto human sequences.

In addition, the human antibodies and binding proteins of the presentinvention are not limited to combinations of VH, VL, CDR or FR regionsthat are themselves found in combination in human antibody molecules.Thus, the human antibodies and binding proteins of the invention caninclude or correspond to combinations of such regions that do notnecessarily exist naturally in humans.

In preferred embodiments, the human antibodies will be fully humanantibodies. “Fully human” antibodies, as used herein, are antibodiescomprising “human” variable region domains and/or CDRs, as definedabove, without substantial non-human antibody sequences or without anynon-human antibody sequences. For example, antibodies comprising humanvariable region domains and/or CDRs “without substantial non-humanantibody sequences” are antibodies, domains and/or CDRs in which only upto 5, 4, 3, 2 or 1 amino acids are amino acids that are not encoded byhuman antibody sequences. Thus, “fully human” antibodies aredistinguished from “humanized” antibodies, which are based onsubstantially non-human variable region domains, e.g., mouse variableregion domains, in which certain amino acids have been changed to bettercorrespond with the amino acids typically present in human antibodies.

The “fully human” antibodies of the invention may be human variableregion domains and/or CDRs without any other substantial antibodysequences, such as being single chain antibodies. Alternatively, the“fully human” antibodies of the invention may be human variable regiondomains and/or CDRs integral with or operatively attached to one or morehuman antibody constant regions. Certain preferred fully humanantibodies are IgG antibodies with the full complement of IgG constantregions.

In other embodiments, “human” antibodies of the invention will bepart-human chimeric antibodies. “Part-human chimeric” antibodies, asused herein, are antibodies comprising “human” variable region domainsand/or CDRs operatively attached to, or grafted onto, a constant regionof a non-human species, such as rat or mouse. Such part-human chimericantibodies may be used, for example, in pre-clinical studies, whereinthe constant region will preferably be of the same species of animalused in the pre-clinical testing. These part-human chimeric antibodiesmay also be used, for example, in ex vivo diagnostics, wherein theconstant region of the non-human species may provide additional optionsfor antibody detection.

The term “fragment” as used herein refers to fragments of biologicalrelevance, e.g., fragments that contribute to antigen binding, e.g.,form part of the antigen binding site, and/or contribute to theinhibition or reduction in function of the CCR4 antigen. Certainpreferred fragments comprise a heavy chain variable region (VH domain)and/or a light chain variable region (VL domain) of the antibodies ofthe invention. Other preferred fragments comprise one or more of theheavy chain CDRs of the antibodies of the invention (or of the VHdomains of the invention), or one or more of the light chain CDRs of theantibodies of the invention (or of the VL domains of the invention).Certain preferred fragments are at least 5 amino acids in length andcomprise at least one CDR region, preferably a CDR3 region, morepreferably a heavy chain CDR3 region.

In embodiments where the antibodies of the invention comprise a fragmentof any of the defined sequences disclosed herein, e.g., are antibodiescomprising VH and/or VL domains of the invention, or are antibodies orbinding proteins comprising one or more CDRs of the invention, thenthese regions/domains are generally separated within the antibody orbinding protein so that each region/domain can perform its biologicalfunction and so that the contribution to antigen binding is retained.Thus, the VH and VL domains are preferably separated by appropriatescaffold sequences/linker sequences and the CDRs are preferablyseparated by appropriate framework regions such as those found innaturally occurring antibodies and/or effective engineered antibodies.Thus, the VH, VL and individual CDR sequences of the invention arepreferably provided within or incorporated into an appropriate frameworkor scaffold to enable antigen binding. Such framework sequences orregions may correspond to naturally occurring framework regions, FR1,FR2, FR3 and/or FR4, as appropriate to form an appropriate scaffold, ormay correspond to consensus framework regions, for example identified bycomparing various naturally occurring framework regions. Alternatively,non-antibody scaffolds or frameworks, e.g., T cell receptor frameworkscan be used.

Appropriate sequences that can be used for framework regions are wellknown and documented in the art and any of these may be used. Preferredsequences for framework regions are one or more (i.e. one, two, three orfour) of the framework regions making up the V_(H) and/or V_(L) domainsof the invention, i.e., one or more of the framework regions disclosedin Tables 1, 2, 3 or 4, or framework regions substantially homologousthereto, and in particular framework regions that allow the maintenanceof antigen specificity, for example framework regions that result insubstantially the same or the same 3D structure of the antibody.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 12, 13, 14 and 15) and/or variable heavy chain (SEQ ID NOs:7, 8, 9, 10), as appropriate, FR regions of SEQ ID NO: 47 (also shown inTable 1), or FR regions substantially homologous thereto, are found inthe antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 12, 13, 14 and 15) and/or variable heavy chain (SEQ ID NOs:7, 8, 16 and 10), as appropriate, FR regions of SEQ ID NO: 48 (alsoshown in Table 2), or FR regions substantially homologous thereto, arefound in the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 12, 13, 14, 15) and/or variable heavy chain (SEQ ID NOs: 7,8, 18 and 10), as appropriate, FR regions of SEQ ID NO: 49 (also shownin Table 3), or FR regions substantially homologous thereto, are foundin the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 12, 13, 14 and 15) and/or variable heavy chain (SEQ ID NOs:19, 8, 9 and 10), as appropriate, FR regions of SEQ ID NO: 50 (alsoshown in Table 4), or FR regions substantially homologous thereto, arefound in the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 21, 13, 23 and 15) and/or variable heavy chain (SEQ ID NOs:19, 8, 9 and 10), as appropriate, FR regions of SEQ ID NO: 51 (alsoshown in Table 5), or FR regions substantially homologous thereto, arefound in the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 12, 13, 23 and 15) and/or variable heavy chain (SEQ ID NOs:19, 8, 9 and 10), as appropriate, FR regions of SEQ ID NO: 52 (alsoshown in Table 6), or FR regions substantially homologous thereto, arefound in the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 21, 13, 23 and 15) and/or variable heavy chain (SEQ ID NOs:7, 8, 9 and 10), as appropriate, FR regions of SEQ ID NO: 53 (also shownin Table 7), or FR regions substantially homologous thereto, are foundin the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 25, 13, 23 and 15) and/or variable heavy chain (SEQ ID NOs:7, 8, 9 and 10), as appropriate, FR regions of SEQ ID NO: 54 (also shownin Table 8), or FR regions substantially homologous thereto, are foundin the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 12, 13, 23 and 15) and/or variable heavy chain (SEQ IDNOs:7, 8, 28 and 10), as appropriate, FR regions of SEQ ID NO: 55 (alsoshown in Table 9), or FR regions substantially homologous thereto, arefound in the antibodies of the invention.

Thus, in the heavy chain FR1 position 26 is preferably E in someembodiments, but G in other embodiments. In the heavy chain FR3 position22 is preferably P in some embodiments, but S in other embodiments andposition 23 is preferably E in some embodiments, but D in otherembodiments. In the light chain FR1 position 7 is preferably P in someembodiments, but Q in other embodiments. In the light chain FR3 position20 is preferably S in some embodiments, but G in other embodiments.

As mentioned above, a preferred homologue of SEQ ID NO: 12 is SEQ ID NO:129 or 130 and a preferred homolog of SEQ ID NO:21 is SEQ ID NO: 131 or132, so all of the statements above should be understood to include areference to SEQ ID NOs 129-132.

In addition, although preferred antibodies of the invention are made upof VH, VL or CDRs of the invention, it should be noted that theantibodies of the invention also encompass one or more VH, VL or CDRs ofthe invention in combination with other VH, VL or CDRs not of theinvention, provided that the CCR4 binding properties or anti-CCR4properties of the antibodies of the invention as outlined herein arestill present.

The term “heavy chain complementarity determining region” (“heavy chainCDR”) as used herein refers to regions of hypervariability within theheavy chain variable region (VH domain) of an antibody molecule. Theheavy chain variable region has three CDRs termed heavy chain CDR1,heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxyterminus. The heavy chain variable region also has four frameworkregions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxyterminus). These framework regions separate the CDRs.

The term “heavy chain variable region” (VH domain) as used herein refersto the variable region of a heavy chain of an antibody molecule.

The term “light chain complementarity determining region” (“light chainCDR”) as used herein refers to regions of hypervariability within thelight chain variable region (VL domain) of an antibody molecule. Lightchain variable regions have three CDRs termed light chain CDR1, lightchain CDR2 and light chain CDR3 from the amino terminus to the carboxyterminus. The light chain variable region also has four frameworkregions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxyterminus). These framework regions separate the CDRs.

The term “light chain variable region” (VL domain) as used herein refersto the variable region of a light chain of an antibody molecule.

It should be noted that the Kabat nomenclature is followed herein, wherenecessary, in order to define the positioning of the CDRs (Kabat et al.,1991, specifically incorporated herein by reference).

A person skilled in the art will appreciate that the proteins andpolypeptides of the invention, such as the light and heavy CDRs, thelight and heavy chain variable regions, antibodies, antibody fragments,and immunoconjugates, may be prepared in any of several ways well knownand described in the art, but are most preferably prepared usingrecombinant methods.

Nucleic acid fragments encoding the light and heavy chain variableregions of the antibodies of the invention can be derived or produced byany appropriate method, e.g., by cloning or synthesis. Such sequencescould, for example, be prepared by cloning appropriate sequences frome.g., human germ line genes and then making any necessary modificationsto the germ line sequences to obtain the sequences of the inventionusing methods well known and described in the art. An alternative andmore efficient method would be to synthesize the appropriate light orheavy chain variable region sequence as overlapping primers, and useprimer extension to obtain the full sequence. This full sequence couldthen be amplified via PCR with primers containing appropriaterestriction sites for further cloning and manipulation, e.g., forcloning into an appropriate expression vector. Five to seven overlappingprimers per variable region are normally be sufficient, thereby makingthis technique very efficient and precise.

Once nucleic acid fragments encoding the light and heavy chain variableregions of the antibodies of the invention have been obtained, thesefragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region fragments intofull length antibody molecules with appropriate constant region domains,or into particular formats of antibody fragment discussed elsewhereherein, e.g., Fab fragments, scFv fragments, etc. Typically, or as partof this further manipulation procedure, the nucleic acid fragmentsencoding the antibody molecules of the invention are generallyincorporated into an appropriate expression vector in order tofacilitate production of the antibodies of the invention.

Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses), so long as the vector iscompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which are operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner that allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vectorcontaining a nucleic acid molecule of the invention, or a fragmentthereof, and the necessary regulatory sequences for the transcriptionand translation of the protein sequence encoded by the nucleic acidmolecule of the invention.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (forexample, see the regulatory sequences described in Goeddel, 1990).Selection of appropriate regulatory sequences is dependent on the hostcell chosen as discussed below, and may be readily accomplished by oneof ordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

The recombinant expression vectors of the invention may also contain aselectable marker gene that facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asneomycin and hygromycin that confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors of the invention and in particular todetermine the effect of a mutation on expression and phenotype. It willbe appreciated that selectable markers can be introduced on a separatevector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes that encode afusion moiety that provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification (for example appropriate “tags” to enablepurification and/or identification may be present, e.g., His tags or myctags). For example, a proteolytic cleavage site may be added to thetarget recombinant protein to allow separation of the recombinantprotein from the fusion moiety subsequent to purification of the fusionprotein. Typical fusion expression vectors include pGEX (Amrad Corp.,Melbourne, Australia), pMal (New England Biolabs, Beverly, Mass.) andpRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to therecombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” and “transfection” are intended toencompass introduction of nucleic acid (e.g., a vector) into a cell byone of many possible techniques known in the art. The term “transformedhost cell” as used herein is intended to also include cells capable ofglycosylation that have been transformed with a recombinant expressionvector of the invention. Prokaryotic cells can be transformed withnucleic acid by, for example, electroporation or calcium-chloridemediated transformation. For example, nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofection, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al., 1989, and other laboratory textbooks.

Suitable host cells include a wide variety of eukaryotic host cells andprokaryotic cells. For example, the proteins of the invention may beexpressed in yeast cells or mammalian cells. Other suitable host cellscan be found in Goeddel, 1990. In addition, the proteins of theinvention may be expressed in prokaryotic cells, such as Escherichiacoli (Zhang et al., 2004).

Yeast and fungi host cells suitable for carrying out the presentinvention include, but are not limited to Saccharomyces cerevisiae, thegenera Pichia or Kluyveromyces and various species of the genusAspergillus. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSecl (Baldari. et al., 1987), pMFa (Kurjan and Herskowitz,1982), pJRY88 (Schultz et al., 1987), and pYES2 (Invitrogen Corporation,San Diego, Calif.). Protocols for the transformation of yeast and fungiare well known to those of ordinary skill in the art (see Hinnen et al.,1978; Ito et al., 1983, and Cullen et al. 1987).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573), NS-1 cells, NS0 (ATCC CRL-11177), and Per.C6® (Crucell,Leiden, Netherlands). Suitable expression vectors for directingexpression in mammalian cells generally include a promoter (e.g.,derived from viral material such as polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40), as well as other transcriptionaland translational control sequences. Examples of mammalian expressionvectors include pCDM8 (Seed, B., 1987) and pMT2PC (Kaufman et al.,1987).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the proteins of the invention may be expressedfrom plant cells (see Sinkar et al., 1987, which reviews the use ofAgrobacterium rhizogenes vectors; see also Zambryski et al., 1984, whichdescribes the use of expression vectors for plant cells, including,among others, PAPS2022, PAPS2023, and PAPS2034).

Insect cells suitable for carrying out the present invention includecells and cell lines from Bombyx, Trichoplusia or Spodotera species.Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., 1983)and the pVL series (Luckow and Summers 1989). Some baculovirus-insectcell expression systems suitable for expression of the recombinantproteins of the invention are described in PCT/US/02442.

Alternatively, the proteins of the invention may also be expressed innon-human transgenic animals such as, rats, rabbits, sheep and pigs(Hammer et al. 1985; Palmiter et al. 1983; Brinster et al. 1985;Palmiter and Brinster 1985, and U.S. Pat. No. 4,736,866).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield (1964); Frische et al., 1996) or synthesisin homogenous solution.

N-terminal or C-terminal fusion proteins comprising the antibodies andproteins of the invention conjugated to other molecules, such asproteins, may be prepared by fusing through recombinant techniques. Theresultant fusion proteins contain an antibody or protein of theinvention fused to the selected protein or marker protein, or tagprotein as described herein. The antibodies and proteins of theinvention may also be conjugated to other proteins by known techniques.For example, the proteins may be coupled using heterobifunctionalthiol-containing linkers as described in WO 90/10457,N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5thioacetate. Examples of proteins that may be used to prepare fusionproteins or conjugates include cell binding proteins such asimmunoglobulins, hormones, growth factors, lectins, insulin, low densitylipoprotein, glucagon, endorphins, transferrin, bombesin,asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA),and truncated myc.

Irrespective of the manner of preparation of a first anti-CCR4 antibodynucleic acid segment, further suitable antibody nucleic acid segmentsmay be readily prepared by standard molecular biological techniques. Inorder to confirm that any variant, mutant or second generation anti-CCR4antibody nucleic acid segment is suitable for use in the presentinvention, the nucleic acid segment will be tested to confirm expressionof an anti-CCR4 antibody in accordance with the present invention.Preferably, the variant, mutant or second generation nucleic acidsegment will also be tested to confirm hybridization under standard,more preferably, standard stringent hybridization conditions. Exemplarysuitable hybridization conditions include hybridization in about 7%sodium dodecyl sulfate (SDS), about 0.5 M NaPO4, about 1 mM EDTA atabout 50° C.; and washing with about 1% SDS at about 42° C.

As a variety of antibodies may be readily prepared, the treatmentmethods of the invention may be executed by providing to the animal orpatient at least a first nucleic acid segment or molecule that expressesa biologically effective amount of at least a first anti-CCR4 antibodyof the invention in the patient. The “nucleic acid segment or moleculethat expresses an anti-CCR4 antibody” will generally be in the form ofat least an expression construct or vector, and may be in the form of anexpression construct or vector comprised within a virus or within arecombinant host cell. Preferred gene therapy vectors of the presentinvention will generally be viral vectors, such as comprised within arecombinant retrovirus, herpes simplex virus (HSV), adenovirus,adeno-associated virus (AAV), cytomegalovirus (CMV), and the like.

A yet further aspect provides an expression construct or expressionvector comprising one or more of the nucleic acid segments or moleculesof the invention. Preferably the expression constructs or vectors arerecombinant. Preferably said constructs or vectors further comprise thenecessary regulatory sequences for the transcription and translation ofthe protein sequence encoded by the nucleic acid molecule of theinvention.

A yet further aspect provides a host cell or virus comprising one ormore expression constructs or expression vectors of the invention. Alsoprovided are host cells or viruses comprising one or more of the nucleicacid molecules of the invention. A host cell or virus expressing anantibody of the invention forms a yet further aspect.

A yet further aspect of the invention provides a method of producing anantibody of the present invention comprising a step of culturing thehost cells of the invention. Preferred methods comprise the steps of (i)culturing a host cell comprising one or more of the recombinantexpression vectors or one or more of the nucleic acid sequences of theinvention under conditions suitable for the expression of the encodedantibody or protein; and optionally (ii) isolating or obtaining theantibody or protein from the host cell or from the growthmedium/supernatant. Such methods of production may also comprise a stepof purification of the antibody or protein product and/or formulatingthe antibody or product into a composition including at least oneadditional component, such as a pharmaceutically acceptable carrier orexcipient.

In embodiments when the antibody or protein of the invention is made upof more than one polypeptide chain (e.g., certain fragments such as Fabfragments), then all the polypeptides are preferably expressed in thehost cell, either from the same or a different expression vector, sothat the complete proteins, e.g., binding proteins of the invention, canassemble in the host cell and be isolated or purified therefrom.

The antibodies of the invention may also be used to produce furtherantibodies that bind to CCR4. Such uses involve for example theaddition, deletion, substitution or insertion of one or more amino acidsin the amino acid sequence of a parent antibody to form a new antibody,wherein said parent antibody is one of the antibodies of the inventionas defined elsewhere herein, and testing the resulting new antibody toidentify antibodies that bind to CCR4. Such methods can be used to formmultiple new antibodies that can all be tested for their ability to bindCCR4. Preferably said addition, deletion, substitution or insertion ofone or more amino acids takes place in one or more of the CDR domains.

Such modification or mutation to a parent antibody can be carried out inany appropriate manner using techniques well known and documented in theart, for example by carrying out methods of random or directedmutagenesis. If directed mutagenesis is to be used then one strategy toidentify appropriate residues for mutagenesis utilizes the resolution ofthe crystal structure of the binding protein-antigen complex, e.g., theAb-Ag complex, to identify the key residues involved in the antigenbinding (Davies and Cohen, 1996). Subsequently, those residues can bemutated to enhance the interaction. Alternatively, one or more aminoacid residues can simply be targeted for directed mutagenesis and theeffect on binding to CCR4 assessed.

Random mutagenesis can be carried out in any appropriate way, e.g., byerror-prone PCR, chain shuffling or mutator E. coli strains.

Thus, one or more of the VH domains of the invention can be combinedwith a single VL domain or a repertoire of VL domains from anyappropriate source and the resulting new antibodies tested to identifyantibodies specific for CCR4. Conversely, one or more of the VL domainsof the invention can be combined with a single VH domain or repertoireof VH domains from any appropriate source and the resulting newantibodies tested to identify antibodies that bind to CCR4.

Similarly, one or more, or preferably all three CDRs of the VH and/or VLdomains of the invention can be grafted into a single VH and/or VLdomain or a repertoire of VH and/or VL domains, as appropriate, and theresulting new antibodies tested to identify antibodies that bind toCCR4.

The targeted mutations of the CDRs, especially CDR3 of the light and/orheavy chains, have been shown to be an effective technique forincreasing antibody affinity and are preferred. Preferably, blocks of 3to 4 amino acids of the CDR3 or specific regions called “hot-spots” aretargeted for mutagenesis.

“Hot spots” are the sequences where somatic hypermutation takes place invivo (and below Neuberger and Milstein, 1995). The hotspot sequences canbe defined as consensus nucleotide sequences in certain codons. Theconsensus sequence is the tetranucleotide, RGYW, in which R can beeither A or G, Y can be C or T and W can be either A or T (Neuberger andMilstein, 1995). In addition, the serine residues encoded by thenucleotides AGY are predominantly present in the CDRs regions of thevariable domain over those encoded by TCN corresponding to a potentialhot-spot sequences (Wagner et al., 1995).

Thus, the nucleotide sequence of the CDRs of the heavy and light chainsof each antibody of the invention can be scanned for the presence of thehot-spot sequences and AGY codons. The identified hot-spots of the CDRregions of the light and heavy chain can then optionally be compared tothe germinal sequences of the heavy and light chains using theInternational ImMunoGen Tics database (IMGT,http://imgt.cines.fr/textes/vquest/) (Davies et al., 1990). A sequence,identical to the germ line, suggest that somatic mutation has notoccurred; therefore random mutations can be introduced mimicking thesomatic events occurring in vivo or alternatively, site directedmutagenesis can be carried out, e.g., at the hot spots and/or AGYcodons. In contrast, a different sequence shows that some somaticmutations have already occurred. It will remain to be determined if thein vivo somatic mutation was optimal.

Preferred hot-spots for mutation are those that code for exposed aminoacids and preferably those that encode amino acids that form part of theantigen binding sites. Other preferred hot-spots for mutation are thosethat code for non-conserved amino acids. The hot-spots that code forburied or conserved amino acids within the CDRs are preferably notmutagenized. These residues are usually critical for the overallstructure and are unlikely to interact with the antigen since they areburied.

Methods of carrying out the above described manipulation of amino acidsand protein domains are well known to a person skilled in the art. Forexample, said manipulations could conveniently be carried out by geneticengineering at the nucleic acid level wherein nucleic acid moleculesencoding appropriate binding proteins and domains thereof are modifiedsuch that the amino acid sequence of the resulting expressed protein isin turn modified in the appropriate way.

Testing the ability of one or more antibodies to specifically bind toCCR4 can be carried out by any appropriate method, which are well knownand described in the art. CCR4+ cell lines may be obtained from culturecollections, or they may be prepared by transforming CCR4-negative cellswith a construct that allows expression of recombinant CCR4. Such cells,or immobilised CCR4 can readily be used to assay binding, for example byconventional methods such as ELISA, BiaCon, etc.

The new antibodies produced by these methods will preferably haveimproved functional properties, e.g. a higher or enhanced affinity (orat least an equivalent affinity) for CCR4 as the parent antibodies, andcan be treated and used in the same way as the antibodies of theinvention as described elsewhere herein (e.g., for therapy, diagnosis,in compositions etc). Alternatively, or additionally, the new antibodieswill have one or more other improved functional properties as describedelsewhere herein.

New antibodies produced, obtained or obtainable by these methods form ayet further aspect of the invention.

This invention further provides compositions comprising at least onehuman antibody or antibody fragment of the invention, optionallyincluding a diluent. Such compositions may be pharmaceuticallyacceptable compositions or compositions for use in laboratory studies.In terms of the pharmaceutical compositions, they may preferably beformulated for parenteral, intravenous or even subcutaneousadministration.

The present invention provides a number of methods and uses of the humanantibodies and antibody fragments of the invention. Concerning allmethods, the terms “a” and “an” are used to mean “at least one”, “atleast a first”, “one or more” or “a plurality” of steps in the recitedmethods, except where specifically stated. This is particularly relevantto the administration steps in the treatment methods. Thus, not only maydifferent doses be employed with the present invention, but differentnumbers of doses, e.g., injections, may be used, up to and includingmultiple injections. Combined therapeutics may be used, administeredbefore, after or during administration of the anti-CCR4 therapeuticantibody.

Various useful in vitro methods and uses of the antibodies orimmunoconjugates of the invention are provided that have importantbiological implications. First provided are methods of, and uses in,binding CCR4, which generally comprise effectively contacting acomposition comprising CCR4 with at least a first anti-CCR4 antibody ofthe invention, or antigen-binding fragment thereof. The antibodies ofthe invention, or immunoconjugates thereof, can thus be used in bindingassays. Suitably useful binding assays include those commonly employedin the art, such as in immunoblots, Western blots, dot blots, RIAs,ELISAs, immunohistochemistry, fluorescent activated cell sorting (FACS),immunoprecipitation, affinity chromatography, and the like.

Methods of, and uses in, detecting CCR4 are provided, which generallycomprise contacting a composition suspected of containing CCR4 or knownto contain CCR4 with at least a first antibody or immunoconjugate of theinvention, or antigen-binding fragment thereof, under conditionseffective to allow the formation of CCR4/antibody complexes anddetecting the complexes so formed. The detection methods and uses may beused in connection with biological samples, e.g., in diagnostics fortumours, and diagnostic kits based thereon are also provided.

The antibody of the invention may also be used to determine whether asubject may benefit from anti-CCR4 therapy. Thus, there is provided theuse of an antibody of the invention for detecting the presence ormeasuring the amount of CCR4 expressed by a cell, target site, tissue ororgan of a subject suffering from a CCR4-related disorder, wherein anincreased presence or amount of CCR4 detected or measured compared to ahealthy control indicates that said subject may benefit from anti-CCR4therapy. Alternatively viewed, there is provided a method of determiningwhether a subject may benefit from anti-CCR4 therapy, comprisingadministering an effective amount of an antibody of the invention to asubject and detecting the presence or measuring the amount of CCR4expressed by a cell, target site, tissue or organ of said subject,wherein an increased presence or amount of CCR4 detected or measuredcompared to a healthy control indicates that said subject may benefitfrom anti-CCR4 therapy.

Said cell or target site may preferably be a solid tumour or ahaematological tumour. Preferably, the presence or amount of CCR4detected or measured is used to predict whether the CCR4-relateddisorder will be susceptible treatment with an anti-CCR4 agent,preferably the anti-CCR4 antibody of the invention. Preferably, thepresence or amount of CCR4 detected or measured is used to decide toadminister an anti-CCR4 agent, preferably the anti-CCR4 antibody of theinvention.

The methods and uses of the present invention are particularly intendedfor use in animals and patients that have, or are at risk fordeveloping, any disease or condition associated with CCR4 expression oractivity or in which CCR4 plays a biological role. Such diseases anddisorders include diseases which are mediated by CCR4 positive cells,typically CCR4+ Th2 or Th17 cells, which, upon binding of a ligand toCCR4, may take part in a signaling pathway which will cause orcontribute to a disorder or disease. They also include diseases causedby aberrant proliferation of cells expressing CCR4. Such aberrantlyproliferating cells may naturally be CCR4+, or they may havemutated/been transformed to express CCR4 As mentioned above, expressionof CCR4 may help cancer cells expressing this antigen to evade theimmune system. Thus, there is provided a method of treating a disease ordisorder mediated by CCR4 and/or characterised by aberrant proliferationof CCR4-positive cells.

Alternatively viewed, there is provided the treatment of a conditionwhich can benefit from one or more of the following

(i) the selective elimination of CCR4+ cells

(ii) the inhibition of CCR4 binding to one or more of its ligands

(iii) the inhibition of CCR4-mediated cellular responses to a CCR4ligand, particularly the inhibition of chemotaxis or increasedintracellular calcium ion concentration (cell activation).

Preferably, the CCR4 ligand is MDC and/or TARC.

It is well known to those of ordinary skill in the art that as CCR4 isinvolved in a wide range of diseases and disorders, a given anti-CCR4therapy, once shown to be effective in any acceptable model system, canbe used to treat the entire range of diseases and disorders connectedwith CCR4 expression.

In one embodiment, the CCR4-mediated condition is a T-helper cell type2-mediated immune disease. By “T-helper cell type 2-mediated immunedisease” is meant a disease involving immunoglobulin E (IgE) and mastcells due to the development and activation of allergen-specific Th2cells.

The CCR4-mediated disease or disorder may be a disease or conditionassociated with inflammation, infection and/or cancer, includinghematological and non-hematological cancers. Such diseases or disorderscan be treated or prevented with the present antibodies andcompositions. Preferred diseases or conditions include: (1) allergicdiseases such as systemic anaphylaxis or hypersensitivity responses,drug allergies, allergic bronchopulmonary aspergillosis (ABPA), insectsting allergies and food allergies, (2) inflammatory bowel diseases,such as Crohn's disease, ulcerative colitis, ileitis and enteritis, (3)vaginitis, (4) psoriasis and inflammatory dermatoses such as dermatitis,eczema, atopic dermatitis, allergic contact dermatitis, urticaria andpruritus, (5) vasculitis, (6) spondyloarthropathies, (7) scleroderma,(8) asthma and respiratory allergic diseases such as allergic asthma,allergic rhinitis, chronic obstructive pulmonary disease,hypersensitivity lung diseases and the like, (9) autoimmune diseases,such as arthritis (including rheumatoid and psoriatic), multiplesclerosis, systemic lupus erythematosus, type I diabetes,glomerulonephritis, and the like, (10) graft rejection (includingallograft rejection and graft-v-host disease), and (11) other diseasesin which undesired inflammatory responses are to be inhibited, such asatherosclerosis, myositis, T-cell mediated neurodegenerative diseases,multiple sclerosis, encephalitis, meningitis, hepatitis, nephritis,sepsis, sarcoidosis, allergic conjunctivitis, otitis, Castleman'sdisease, sinusitis, LPS-induced endotoxic shock, Behcet's syndrome andgout, (12) cancers, both hematological and non-hematological cancers,preferably Breast cancer, Colorectal cancer, Esophageal cancer, Gastriccancer, Hepatocellular carcinoma, Lung cancer, Melanoma, Ovarian cancer,Pancreatic cancer, Adult T-cell leukemia/lymphoma (ATL), PeripheralT-cell lymphoma, unspecified Diffuse large B-cell lymphoma, Hodgkin'slymphoma, B-cell chronic lymphocytic leukemia, CTCL, particularlyMycosis fungoides, Sézary syndrome, cervical cancer, kidney cancer,brain cancer, prostate cancer, stomach cancer (13) infections such asEpstein-Barr virus (EBV) infection, HIV infection and other viralinfections.

The binding of the antibody of the present invention to CCR4 may alsoimpair the ability of CCR4-positive aberrant cells such as cancer cellsto evade the host immune system. The antibodies of the present inventionmay be used to block the suppression of DCs by Treg cells, so there isprovided the use of an anti-CCR4 antibody of the present invention as anadjuvant in a vaccine. The vaccine is preferably a vaccine for cancer oran infectious disease. The vaccine may be a preventative vaccine or acurative vaccine. By “adjuvant” is meant an agent which enhances theimmune response of a host to an antigen. When used as vaccine adjuvants,the antibodies of the present invention are therefore typicallyadministered in conjunction or combined with an antigen against which itis desired to elicit an immune response.

As used herein, the term “aberrant proliferation” means cellproliferation that deviates from the normal, proper, or expected course.For example, aberrant cell proliferation may include inappropriateproliferation of cells whose DNA or other cellular components havebecome damaged or defective. Aberrant cell proliferation may includecell proliferation whose characteristics are associated with anindication caused by, mediated by, or resulting in inappropriately highlevels of cell division, inappropriately low levels of apoptosis, orboth. Such indications may be characterized, for example, by single ormultiple local abnormal proliferations of cells, groups of cells, ortissue (s), cancerous or non-cancerous, benign or malignant.

Any reference to “tumour(s)” herein also refers to “cancer(s)” or“carcinoma(s)”. Metastatic cancers can also be treated, as can thereduction of metastases from a primary tumour. So-called minimalresidual disease (MRD), which is left in post-surgery patients, may beamenable for immunotherapy with anti-CCR4 antibodies.

The present invention thus further provides methods of, and uses in,treating a disease as defined above, comprising administering to ananimal or patient with such a disease, a therapeutically effectiveamount of an anti-CCR4 antibody of the invention, or an antigen-bindingfragment or immunoconjugate of such an anti-CCR4 antibody.

A yet further aspect of the invention provides the use of the antibodiesof the invention or an antigen-binding fragment or immunoconjugate ofsuch an antibody in the manufacture of a composition or medicament foruse in therapy, imaging or diagnosis.

A yet further aspect provides the antibodies of the invention or anantigen-binding fragment or immunoconjugate of such an antibody for usein therapy, diagnosis or imaging.

In addition, the invention provides compositions comprising theantibodies of the invention or an antigen-binding fragment orimmunoconjugate of such an antibody with one or more pharmaceuticallyacceptable excipient, carrier, diluent, buffer or stabilizer.

The in vivo methods as described herein are generally carried out in amammal. Any mammal may be treated, for example humans and any livestock,domestic or laboratory animal. Specific examples include mice, rats,pigs, cats, dogs, sheep, rabbits, cows and monkey. Preferably, however,the mammal is a human.

Thus, the term “animal” or “patient” as used herein includes any mammal,for example humans and any livestock, domestic or laboratory animal.Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits,cows and monkey. Preferably, however, the animal or patient is a humansubject.

This invention links both methods of treating disorders as defined aboveusing unconjugated or naked antibodies and fragments thereof, and CCR4+cell, preferably CCR4+ tumour cell, targeting methods usingimmunoconjugates in which an antibody of the invention orantigen-binding fragment thereof, is operatively attached to atherapeutic agent. Unless otherwise specifically stated or made clear inscientific terms, the terms “antibody and fragment thereof”, as usedherein, therefore mean an “unconjugated or naked” antibody or fragment,which is not attached to another agent, particularly a therapeutic ordiagnostic agent. These definitions do not exclude modifications of theantibody, such as, by way of example only, modifications to improve thebiological half life, affinity, avidity or other properties of theantibody, or combinations of the antibody with other effectors.

The treatment methods and uses of the invention also encompass the useof both unconjugated or naked antibodies and immunoconjugates. In theimmunoconjugate-based treatment methods, an antibody of the invention,or antigen-binding fragment thereof, is preferably operatively attachedto a second therapeutic agent (the anti-CCR4 antibody itself, being thefirst therapeutic agent). The therapeutic agent may for example be ananti-cancer agent or an anti-inflammatory agent, includingcorticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs).

The foregoing treatment methods and uses will generally involve theadministration of the pharmaceutically effective composition to theanimal or patient systemically, such as by transdermal, intramuscular,intravenous injection and the like. However, any route of administrationthat allows the therapeutic agent to localize to the tumour site orsites, will be acceptable. Therefore, other suitable routes of deliveryinclude oral, nasal or respiratory and topical.

“Administration”, as used herein, means provision or delivery ofanti-CCR4 antibody therapeutics in an amount(s) and for a period oftime(s) effective to exert therapeutic, e.g. anti-tumour effects. Thepassive administration of proteinaceous therapeutics is generallypreferred, in part, for its simplicity and reproducibility.

However, the term “administration” is herein used to refer to any andall means by which anti-CCR4 antibodies of the invention are deliveredor otherwise provided to the target site. “Administration” thereforeincludes the provision of cells that produce the anti-CCR4 antibody ofthe invention in a manner effective to result in delivery to the targetsite. In such embodiments, it may be desirable to formulate or packagethe cells in a selectively permeable membrane, structure or implantabledevice, generally one that can be removed to cease therapy. Exogenousanti-CCR4 antibody of the invention will still generally be preferred,as this represents a non-invasive method that allows the dose to beclosely monitored and controlled.

The therapeutic methods and uses of the invention also extend to theprovision of nucleic acids that encode an anti-CCR4 antibody of theinvention in a manner effective to result in their expression in thevicinity of the tumour or their localization to the target site. Anygene therapy technique may be employed, such as naked DNA delivery,recombinant genes and vectors, cell-based delivery, including ex vivomanipulation of patients' cells, and the like.

The anti-CCR4 antibodies of the invention can also be used to deliverother therapeutic or diagnostic agents to the target site In suchembodiments, the other therapeutic or diagnostic agents are generallyoperatively attached to the anti-CCR4 antibodies of the invention.

The “therapeutically effective amounts” for use in the invention areamounts of anti-CCR4 antibody of the invention, or immunoconjugatesthereof, effective to specifically kill at least a portion of targetCCR4+ cells; to specifically induce apoptosis in at least a portion oftarget CCR4+ cells; to specifically induce necrosis in at least aportion of target CCR4+ cells; to inhibit the binding of a CCR4 ligandto CCR4; to inhibit CCR4-mediated cellular responses to a CCR4 ligand,preferably inhibit the increase in intracellular calcium ionconcentration in response to a CCR4 ligand; to reduce inflammation;and/or to induce tumour regression or remission upon administration toanimals or patients having a CCR4+ tumour. Such effects are preferablyachieved while exhibiting little or no binding to, or little or nokilling of cells in normal, healthy tissues; and exerting negligible ormanageable adverse side effects on normal, healthy tissues of the animalor patient.

By “target site” is meant the location of CCR4+ cells which mediate adisorder or which proliferate in an aberrant manner causing orexacerbating a disorder. The target site may thus for example be atumour or the site of CCR4-mediated inflammation. “Target cells” areCCR4+ cells which mediate a disorder or which proliferate in an aberrantmanner causing or exacerbating a disorder. Thus, target cells may forexample include CCR4+ tumour cells, CCR4+ Treg cells and/or CCR4+ Th2cells.

The terms “preferentially” and “specifically”, as used herein in thecontext of killing or inducing apoptosis or of inducing necrosis ofCCR4+ cells such as CCR4+ tumour cells or of reducing inflammation or ofinducing tumour regression or remission, thus mean that the anti-CCR4antibody of the invention or immunoconjugates thereof, function toachieve CCR4+ target cell destruction, e.g. tumour cell destructionand/or tumour necrosis, that is substantially confined to the targetsite, and does not substantially extend to causing destruction and/ortissue necrosis in normal, healthy tissues of the animal or subject.

Anti-CCR4 antibodies of the invention or therapeutic conjugates arepreferably linked to one or more radiotherapeutic agents,chemotherapeutic agents, anti-angiogenic agents, apoptosis-inducingagents, anti-tubulin drugs, anti-cellular or cytotoxic agents, cytokineor chemokine antagonists, inhibitors of cytokine or chemokineexpression, ATPase inhibitors, anti-inflammatory agents, otherantibodies (e.g. as bispecific antibodies) or coagulants (coagulationfactors) or anti-inflammatory agents such as corticosteroids, preferablyglucocorticoids, or non-steroidal anti-inflammatory drugs (NSAIDs).

The invention thus provides a range of conjugated antibodies andfragments thereof in which the anti-CCR4 antibody is operativelyattached to at least one other therapeutic or diagnostic agent. The term“immunoconjugate” is broadly used to define the operative association ofthe antibody with another effective agent and is not intended to refersolely to any type of operative association, and is particularly notlimited to chemical “conjugation”. Recombinant fusion proteins areparticularly contemplated. So long as the delivery or targeting agent isable to bind to the target and the therapeutic or diagnostic agent issufficiently functional upon delivery, the mode of attachment will besuitable.

Attachment of agents via the carbohydrate moieties on antibodies is alsocontemplated. Glycosylation, both O-linked and N-linked, naturallyoccurs in antibodies. Recombinant antibodies can be modified to recreateor create additional glycosylation sites if desired, which is simplyachieved by engineering the appropriate amino acid sequences (such asAsn-X-Ser, Asn-X-Thr, Ser, or Thr where X is any amino acid except Pro)into the primary sequence of the antibody.

Currently preferred agents for use in anti-CCR4 antibody or therapeuticconjugates of the invention and related methods and uses are those thatcomplement or enhance the effects of the antibody and/or those selectedfor a particular type of disorder (e.g. tumour type) or patient.

“Therapeutic agents that complement or enhance the effects of theantibody” include radiotherapeutic agents, chemotherapeutic agents,anti-angiogenic agents, apoptosis-inducing agents, anti-tubulin drugs,anti-cellular or cytotoxic agents, coagulants, cytokine or chemokineantagonists, inhibitors of cytokine or chemokine expression, ATPaseinhibitors, anti-inflammatory agents such as corticosteroids, preferablyglucocorticoids, or non-steroidal anti-inflammatory drugs (NSAIDs),other antibodies, (e.g. as bispecific antibodies), any one or more ofwhich are preferred for use herewith.

Currently preferred anti-cancer, particularly anti-leukaemia agentsinclude Anthracycline drugs such as daunorubicin, Doxorubicin,Cytarabine, 6-thioguanine,

Mitoxantrone, busulfan (Myleran®), dasatinib (Sprycel™), prednisone,vincristine sulfate (Oncovin®), Chlorambucil, Fludarabine, Pentostatinand Cladribine.

Currently preferred agents for the treatment of ATL include zidovudine(azidothymidine) and the CHOP regimen. CHOP stands for Cyclophosphamide,Hydroxydaunorubicin (Adriamycin), Oncovin (Vincristine),Prednisone/Prednisolone.

Currently preferred anti-angiogenic agents include angiostatin,endostatin, any one of the angiopoietins, vasculostatin, canstatin andmaspin.

“Anti-tubulin drug(s)”, as used herein, means any agent, drug, prodrugor combination thereof that inhibits cell mitosis, preferably bydirectly or indirectly inhibiting tubulin activities necessary for cellmitosis, preferably tubulin polymerization or depolymerization.Currently preferred anti-tubulin drugs include colchicine, taxol,vinblastine, vincristine, vindescine and one or more of thecombretastatins.

Currently preferred NSAIDs include COX-2 inhibitors, sulphonanilides,licofelone and omega-3 fatty acids

The attachment or association of the preferred agents with anti-CCR4antibodies of the invention gives “immunoconjugates”, wherein suchimmunoconjugates often have enhanced and even synergistic therapeuticproperties, e.g. anti-tumour or anti-inflammatory properties.

The use of anti-cellular and cytotoxic agents results in anti-CCR4antibody “immunotoxins” of the invention, whereas the use of coagulationfactors results in anti-CCR4 antibody “coaguligands” of the invention.

The use of at least two therapeutic agents is also contemplated, such ascombinations of one or more radiotherapeutic agents, chemotherapeuticagents, anti-angiogenic agents, apoptosis-inducing agents, anti-tubulindrugs, anti-cellular and cytotoxic agents, cytokine or chemokineantagonists, inhibitors of cytokine or chemokine expression, ATPaseinhibitors, anti-inflammatory agents such as corticosteroids, preferablyglucocorticoids, or non-steroidal anti-inflammatory drugs (NSAIDs),other antibodies, (e.g. as bispecific antibodies) and coagulationfactors.

In certain applications, the anti-CCR4 antibody therapeutics of theinvention will be operatively attached to cytotoxic, cytostatic orotherwise anti-cellular agents that have the ability to kill or suppressthe growth or cell division of cells. Suitable anti-cellular agentsinclude chemotherapeutic agents, as well as cytotoxins and cytostaticagents. Cytostatic agents are generally those that disturb the naturalcell cycle of a target cell, preferably so that the cell is taken out ofthe cell cycle.

Exemplary chemotherapeutic agents include: hormones, such as steroids;anti-metabolites, such as cytosine arabinoside, fluorouracil,methotrexate or aminopterin; anthracyclines; mitomycin C; vincaalkaloids; antibiotics; demecolcine; etoposide; mithramycin; andanti-tumor alkylating agents, such as chlorambucil or melphalan. Certainpreferred anti-cellular agents are DNA synthesis inhibitors, such asdaunorubicin, doxorubicin/adriamycin, and the like. Overall,taxol/paclitaxel, docetaxel, cisplatin, gemcitabine, a combretastatinand doxorubicin/adriamycin are currently preferred anti-cancer agents.

V-type ATPase inhibitors are also currently preferred, such assalicylihalamide, concanamycin or bafilomycin, as are protein synthesisinhibitors, such as psymberin, pederin, irciniastatin A.

In certain therapeutic applications, toxin moieties will be preferred,due to the much greater ability of most toxins to deliver a cell killingeffect, as compared to other potential agents. Therefore, certainpreferred anti-cellular agents for anti-CCR4 antibody constructs of theinvention are plant-, fungus- or bacteria-derived toxins. Exemplarytoxins include epipodophyllotoxins; bacterial endotoxin or the lipid Amoiety of bacterial endotoxin; ribosome inactivating proteins, such assaporin or gelonin; a-sarcin; aspergillin; restrictocin; ribonucleases,such as placental ribonuclease; diphtheria toxin and pseudomonasexotoxin. Currently preferred examples are ricin, gelonin, abrin,diphtheria, pseudomonas and pertussis toxins.

Certain preferred toxins are the A chain toxins, such as ricin A chain.The most preferred toxin moiety is often ricin A chain that has beentreated to modify or remove carbohydrate residues, so called“deglycosylated A chain” (dgA). Deglycosylated ricin A chain ispreferred because of its extreme potency, longer half-life, and becauseit is economically feasible to manufacture it a clinical grade andscale. Recombinant and/or truncated ricin A chain may also be used.

The anti-CCR4 antibody therapeutics of the invention may comprise acomponent that is capable of promoting coagulation, i.e., a coagulant.Here, the targeting antibody may be directly or indirectly, e.g., viaanother antibody, linked to a factor that directly or indirectlystimulates coagulation.

Preferred coagulation factors for such uses are Tissue Factor (TF) andTF derivatives, such as truncated TF (tTF), dimeric, trimeric,polymeric/multimeric TF, and mutant TF deficient in the ability toactivate Factor VII. Other suitable coagulation factors include vitaminK-dependent coagulants, such as Factor II/IIa, Factor VII/VIIa, FactorIX/IXa and Factor X/Xa; vitamin K-dependent coagulation factors thatlack the Gla modification; Russell's viper venom Factor X activator;platelet-activating compounds, such as thromboxane A2 and thromboxane A2synthase; and inhibitors of fibrinolysis, such as α2-antiplasmin.Overall, truncated Tissue Factor (tTF) is currently preferred.

The preparation of immunoconjugates and immunotoxins is generally wellknown in the art (see, e.g., U.S. Pat. No. 4,340,535). Each of thefollowing patents are further incorporated herein by reference for thepurposes of even further supplementing the present teachings regardingimmunotoxin generation, purification and use: U.S. Pat. Nos. 6,004,554;5,855,866; 5,965,132; 5,776,427; 5,863,538; 5,660,827 and 6,051,230.

A variety of chemotherapeutic and other pharmacological agents can alsobe successfully conjugated to anti-CCR4 antibody therapeutics of theinvention. Exemplary antineoplastic agents that have been conjugated toantibodies include doxorubicin, daunomycin, methotrexate andvinblastine. Moreover, the attachment of other agents such asneocarzinostatin, macromycin, trenimon and α-amanitin has been described(see U.S. Pat. Nos. 5,660,827; 5,855,866; and 5,965,132; eachincorporated herein.)

The preparation of coaguligands is also easily practiced. The operableassociation of one or more coagulation factors with an anti-CCR4antibody of the invention may be a direct linkage, such as thosedescribed above for the immunotoxins. Alternatively, the operativeassociation may be an indirect attachment, such as where the antibody isoperatively attached to a second binding region, preferably an antibodyor antigen binding region of an antibody, which binds to the coagulationfactor. The coagulation factor should be attached to the anti-CCR4antibody of the invention at a site distinct from its functionalcoagulating site, particularly where a covalent linkage is used to jointhe molecules.

Bispecific or trispecific antibodies may also be employed in the methodsof the invention. In such antibodies one arm binds to CCR4 and is anantibody of the present invention. Methods for preparing bispecificantibodies are well known and described in the art.

In the preparation of immunoconjugates, immunotoxins and coaguligands,recombinant expression may be employed. The nucleic acid sequencesencoding the chosen anti-CCR4 antibody of the invention, and therapeuticagent, toxin or coagulant, are attached in-frame in an expressionvector. Recombinant expression thus results in translation of thenucleic acid to yield the desired immunoconjugate. Chemicalcross-linkers and avidin:biotin bridges may also join the therapeuticagents to the anti-CCR4 antibody of the invention.

The compositions and methods of the present invention may be used incombination with other therapeutics and diagnostics. In terms ofbiological agents, preferably diagnostic or therapeutic agents, for use“in combination” with an anti-CCR4 antibody in accordance with thepresent invention, the term “in combination” is succinctly used to covera range of embodiments. The “in combination” terminology, unlessotherwise specifically stated or made clear from the scientificterminology, thus applies to various formats of combined compositions,pharmaceuticals, cocktails, kits, methods, and first and second medicaluses.

The “combined” embodiments of the invention thus include, for example,where the anti-CCR4 of the invention is a naked antibody and is used incombination with an agent or therapeutic agent that is not operativelyattached thereto. In such cases, the agent or therapeutic agent may beused in a non-targeted or targeted form. In “non-targeted form”, theagent, particularly therapeutic agents, will generally be used accordingto their standard use in the art. In “targeted form”, the agent willgenerally be operatively attached to a distinct antibody or targetingregion that delivers the agent or therapeutic agent to the targetdisease site. The use of such targeted forms of biological agents, bothdiagnostics and therapeutics, is also quite standard in the art.

In other “combined” embodiments of the invention, the anti-CCR4 antibodyof the invention is an immunoconjugate wherein the antibody is itselfoperatively associated or combined with the agent or therapeutic agent.The operative attachment includes all forms of direct and indirectattachment as described herein and known in the art.

The “combined” uses, particularly in terms of an anti-CCR4 antibody ofthe invention in combination with therapeutic agents, also includecombined compositions, pharmaceuticals, cocktails, kits, methods, andfirst and second medical uses wherein the therapeutic agent is in theform of a prodrug. In such embodiments, the activating component able toconvert the prodrug to the functional form of the drug may again beoperatively associated with the anti-CCR4 antibodies of the presentinvention.

In certain preferred embodiments, the therapeutic compositions,combinations, pharmaceuticals, cocktails, kits, methods, and first andsecond medical uses will be “prodrug combinations”. As will beunderstood by those of ordinary skill in the art, the term “prodrugcombination”, unless otherwise stated, means that the antibody of theinvention is operatively attached to a component capable of convertingthe prodrug to the active drug, not that the antibody is attached to theprodrug itself. However, there is no requirement that the prodrugembodiments of the invention need to be used as prodrug combinations.Accordingly, prodrugs may be used in any manner that they are used inthe art, including in ADEPT and other forms.

Thus, where combined compositions, pharmaceuticals, cocktails, kits,methods, and first and second medical uses are described, preferably interms of diagnostic agents, and more preferably therapeutic agents, thecombinations include anti-CCR4 antibodies that are naked antibodies andimmunoconjugates, and wherein practice of the in vivo embodiments of theinvention involves the prior, simultaneous or subsequent administrationof the naked antibodies or immunoconjugate and the biological,diagnostic or therapeutic agent; so long as, in some conjugated orunconjugated form, the overall provision of some form of the antibodyand some form of the biological, diagnostic or therapeutic agent isachieved.

The foregoing and other explanations of the effects of the presentinvention on tumors are made for simplicity to explain the combined modeof operation, type of attached agent(s) and such like. This descriptiveapproach should not be interpreted as either an understatement or anoversimplification of the beneficial properties of the anti-CCR4antibodies of the invention. It will therefore be understood that suchantibodies themselves have anti-CCR4 properties and thatimmunoconjugates of such antibodies will maintain these properties andcombine them with the properties of the attached agent; and further,that the combined effect of the antibody and any attached agent willtypically be enhanced and/or magnified.

The invention therefore provides compositions, pharmaceuticalcompositions, therapeutic kits and medicinal cocktails comprising,optionally in at least a first composition or container, a biologicallyeffective amount of at least a first anti-CCR4 antibody of theinvention, or an antigen-binding fragment or immunoconjugate of such ananti-CCR4 antibody; and a biologically effective amount of at least asecond biological agent, component or system.

The “at least a second biological agent, component or system” will oftenbe a therapeutic or diagnostic agent, component or system, but it neednot be. For example, the at least a second biological agent, componentor system may comprise components for modification of the antibodyand/or for attaching other agents to the antibody. Certain preferredsecond biological agents, components or systems are prodrugs orcomponents for making and using prodrugs, including components formaking the prodrug itself and components for adapting the antibodies ofthe invention to function in such prodrug or ADEPT embodiments.

Where therapeutic or diagnostic agents are included as the at least asecond biological agent, component or system, such therapeutics and/ordiagnostics will typically be those for use in connection with thetreatment or diagnosis of one or more of the disorders defined above.

Thus, in certain embodiments “at least a second therapeutic agent” willbe included in the therapeutic kit or cocktail. The term “at least asecond therapeutic agent” is chosen in reference to the anti-CCR4antibody of the invention being the first therapeutic agent. Theantibodies of the invention may thus be combined with chemotherapeuticagents, radiotherapeutic agents, cytokine or chemokine antagonists,inhibitors of cytokine or chemokine expression, anti-angiogenic agents,apoptosis-inducing agents or anti-cancer immunotoxins or coaguligands,or anti-inflammatory agents including corticosteroids and NSAIDs, someexamples of which are discussed elsewhere herein.

Other exemplary anti-cancer agent include, e.g., neomycin,podophyllotoxin(s), TNF-α, αvβ3 antagonists, calcium ionophores,calcium-flux inducing agents, and any derivative or prodrug thereof.Currently preferred anti-tubulin drugs include colchicine, taxol,vinblastine, vincristine, vindescine, a combretastatin or a derivativeor prodrug thereof.

In terms of compositions, kits and/or medicaments of the invention, thecombined effective amounts of the therapeutic agents may be comprisedwithin a single container or container means, or comprised withindistinct containers or container means. The cocktails will generally beadmixed together for combined use. Agents formulated for intravenousadministration will often be preferred. Imaging components may also beincluded. The kits may also comprise instructions for using the at leasta first antibody and the one or more other biological agents included.

Speaking generally, the at least a second therapeutic agent may beadministered to the animal or patient substantially simultaneously withthe anti-CCR4 antibody of the invention; such as from a singlepharmaceutical composition or from two pharmaceutical compositionsadministered closely together.

Alternatively, the at least a second therapeutic agent may beadministered to the animal or patient at a time sequential to theadministration of the anti-CCR4 antibody of the invention. “At a timesequential”, as used herein, means “staggered”, such that the at least asecond anti-cancer agent is administered to the animal or patient at atime distinct to the administration of the anti-CCR4 antibody of theinvention. Generally, the two agents are administered at timeseffectively spaced apart to allow the two agents to exert theirrespective therapeutic effects, i.e., they are administered at“biologically effective time intervals”. The at least a secondtherapeutic agent may be administered to the animal or patient at abiologically effective time prior to the anti-CCR4 antibody of theinvention, or at a biologically effective time subsequent to thattherapeutic.

Accordingly, the present invention provides methods for treating ananimal or patient with a tumor, comprising:

(a) subjecting the animal or patient to a first treatment thatsubstantially reduces the tumor burden; and

(b) subsequently administering at least a first anti-CCR4 antibody ofthe invention, or antigen-binding fragment thereof; optionally whereinthe antibody or fragment is operatively associated with a secondtherapeutic agent.

Preferred first treatments include surgical resection andchemotherapeutic intervention.

In other embodiments, the present invention provides methods fortreating an animal or patient with a CCR4-mediated disorder, comprising:

(a) subjecting the animal or patient to a first treatment thatsubstantially reduces the CCR4-mediated burden such as inflammation; and

(b) subsequently administering at least a first anti-CCR4 antibody ofthe invention, or antigen-binding fragment thereof; optionally whereinthe antibody or fragment is operatively associated with a secondtherapeutic agent.

In certain other embodiments, the antibodies and immunoconjugates of theinvention may be combined with one or more diagnostic agents, typicallydiagnostic agents for use in connection with the diagnosis of a disorderas defined above. A range of diagnostic compositions, kits and methodsare thus included within the invention.

Yet further aspects are methods of diagnosis or imaging of a subjectcomprising the administration of an appropriate amount of an antibody orother protein of the invention as defined herein to the subject anddetecting the presence and/or amount and/or the location of the antibodyor other protein of the invention in the subject.

In one embodiment, the invention provides a method of reducingimmunosuppression associated with CCR4 expression in an animal,comprising administering to said animal the antibody of the invention,or an immunoconjugate thereof, in an amount effective to form complexesbetween said antibody and CCR4 in said animal, thereby reducingimmunosuppression associated with CCR4 expression in an animal.

Appropriate diseases to be imaged or diagnosed in accordance with theabove described uses and methods include any disease and preferably anycancer as described elsewhere herein.

In one embodiment, the invention provides a method of diagnosing diseaseor monitoring the progress of disease in an animal comprising the stepof:

(a) contacting a test sample taken from said animal with an antibody ofthe invention or an immunoconjugate thereof.

In a further embodiment, the invention provides a method of diagnosingdisease or monitoring the progress of disease in an animal comprisingthe steps of:

(a) contacting a test sample taken from said animal with an antibody ofthe invention or an immunoconjugate thereof;

(b) measuring or detecting the presence and/or amount and/or location ofantibody-antigen complex in the test sample; and, optionally

(c) comparing the presence and/or amount of antibody-antigen complex inthe test sample to a control.

In the above methods, said contacting step is carried out underconditions that permit the formation of an antibody-antigen complex.Appropriate conditions can readily be determined by a person skilled inthe art.

In the above methods any appropriate test sample may be used, forexample biopsy cells, tissues or organs suspected of being affected bydisease or histological sections.

In certain of the above methods, the presence of any amount ofantibody-antigen complex in the test sample would be indicative of thepresence of disease. Preferably, for a positive diagnosis to be made,the amount of antibody-antigen complex in the test sample is greaterthan, preferably significantly greater than, the amount found in anappropriate control sample. More preferably, the significantly greaterlevels are statistically significant, preferably with a probabilityvalue of <0.05. Appropriate methods of determining statisticalsignificance are well known and documented in the art and any of thesemay be used.

Monitoring the progress of a disease may also involve monitoring thepresence and/or amount of antibody-antigen complex in test samples overtime. Thus, monitoring may involve (d) comparing the presence and/oramount of antibody-antigen complex in a first test sample to thepresence and/or amount of antibody-antigen complex in a second testsample taken from said animal. By “first test sample” is meant a samplethat was taken prior to taking the “second test sample”, for example 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days, weeks, months or years priorto taking the second test sample. A decrease in the amount ofantibody-antigen complex in the second test sample compared to the firsttest sample is indicative of the disease regressing, whereas an increaseis indicative of the disease progressing.

Appropriate control samples could be readily chosen by a person skilledin the art, for example, in the case of diagnosis of a particulardisease, an appropriate control would be a sample from a subject thatdid not have that disease. Appropriate control “values” could also bereadily determined without running a control “sample” in every test,e.g., by reference to the range for normal subjects known in the art.

For use in the diagnostic or imaging applications, the antibodies of theinvention may be labeled with a detectable marker such as a radio-opaqueor radioisotope, such as 3H, 14C, 32P, 35S, 1231, 1251, 1311; aradioactive emitter (e.g., α, β or γ emitters); a fluorescent(fluorophore) or chemiluminescent (chromophore) compound, such asfluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such asalkaline phosphatase, beta-galactosidase or horseradish peroxidase; animaging agent; or a metal ion; or a chemical moiety such as biotin whichmay be detected by binding to a specific cognate detectable moiety,e.g., labelled avidin/streptavidin. Methods of attaching a label to abinding protein, such as an antibody or antibody fragment, are known inthe art. Such detectable markers allow the presence, amount or locationof binding protein-antigen complexes in the test sample to be examined.

Preferred detectable markers for in vivo use include an X-ray detectablecompound, such as bismuth (III), gold (III), lanthanum (III) or lead(II); a radioactive ion, such as copper67, gallium67, gallium68,indium111, indium113, iodine123, iodine125, iodine131, mercuryl97,mercury203, rhenium186, rhenium188, rubidium97, rubidium103,technetium99m or yttrium90; a nuclear magnetic spin-resonance isotope,such as cobalt (II), copper (II), chromium (III), dysprosium (III),erbium (III), gadolinium (III), holmium (III), iron (II), iron (III),manganese (II), neodymium (III), nickel (II), samarium (III), terbium(III), vanadium (II) or ytterbium (III); or rhodamine or fluorescein.

The invention also includes diagnostic or imaging agents comprising theantibodies of the invention attached to a label that produces adetectable signal, directly or indirectly. Appropriate labels aredescribed elsewhere herein.

The invention further includes kits comprising one or more of theantibodies, immunoconjugates or compositions of the invention or one ormore of the nucleic acid molecules encoding the antibodies of theinvention, or one or more recombinant expression vectors comprising thenucleic acid sequences of the invention, or one or more host cells orviruses comprising the recombinant expression vectors or nucleic acidsequences of the invention. Preferably said kits are for use in themethods and uses as described herein, e.g., the therapeutic, diagnosticor imaging methods as described herein, or are for use in the in vitroassays or methods as described herein. The antibody in such kits maypreferably be an antibody conjugate as described elsewhere herein, e.g.,may be conjugated to a detectable moiety or may be an immumoconjugate.Preferably said kits comprise instructions for use of the kitcomponents, for example in diagnosis. Preferably said kits are fordiagnosing or treating diseases as described elsewhere herein andoptionally comprise instructions for use of the kit components todiagnose or treat such diseases.

The antibodies of the invention as defined herein may also be used asmolecular tools for in vitro or in vivo applications and assays. As theantibodies have an antigen binding site, these can function as membersof specific binding pairs and these molecules can be used in any assaywhere the particular binding pair member is required.

Thus, yet further aspects of the invention provide a reagent thatcomprises an antibody of the invention as defined herein and the use ofsuch antibodies as molecular tools, for example in in vitro or in vivoassays.

Cancer treatment may also be carried out by:

(a) forming an image of a tumor by administering to an animal or patienthaving a tumor a diagnostic amount of at least a firstdetectably-labeled anti-CCR4 antibody of the invention, comprising adiagnostic agent operatively attached to the anti-CCR4 antibody of theinvention, thereby forming a detectable image of the tumor; and

(b) subsequently administering to the same animal or patient atherapeutically optimized amount of at least a first naked anti-CCR4antibody of the invention or therapeutic agent-antibody construct usingsuch an antibody, thereby causing an anti-tumor effect.

SEQ ID NOs: 16 and 18 are identical, so in any of the embodimentsdisclosed herein, a reference to SEQ ID NO:16 should be understood toinclude a reference to SEQ ID NO: 18 and vice versa.

The invention will now be described in more detail in the followingnon-limited examples with reference to the Tables and Figures in which:

Table 1 lists some of the sequences disclosed herein relating toantibody 208.

Table 2 lists some of the sequences disclosed herein relating toantibody 306.

Table 3 lists some of the sequences disclosed herein relating toantibody 308.

Table 4 lists some of the sequences disclosed herein relating toantibody 406.

Table 5 lists some of the sequences disclosed herein relating toantibody 501.

Table 6 lists some of the sequences disclosed herein relating toantibody 503.

Table 7 lists some of the sequences disclosed herein relating toantibody 601.

Table 8 lists some of the sequences disclosed herein relating toantibody 603.

Table 9 lists some of the sequences disclosed herein relating toantibody 803.

Table 10 lists some of the sequences disclosed herein relating to theIgG form of antibody 208. The variable regions are underlined.

Table 11 lists some of the sequences disclosed herein relating to IgGform of antibody 306. The variable regions are underlined.

Table 12 lists some of the sequences disclosed herein relating to IgGform of antibody 308. The variable regions are underlined.

Table 13 lists some of the sequences disclosed herein relating to IgGform of antibody 406. The variable regions are underlined.

Table 14 lists some of the sequences disclosed herein relating to IgGform of antibody 501. The variable regions are underlined.

Table 15 lists some of the sequences disclosed herein relating to IgGform of antibody 503. The variable regions are underlined.

Table 16 lists some of the sequences disclosed herein relating to IgGform of antibody 601. The variable regions are underlined.

Table 17 lists some of the sequences disclosed herein relating to IgGform of antibody 603. The variable regions are underlined.

Table 18 lists some of the sequences disclosed herein relating to IgGform of antibody 803. The variable regions are underlined.

Table 19 shows calculated IC50 values, derived from TARC-mediatedCa-Flux inhibition.

Table 20 shows determined IC50 values from ligand-interfering bindingexperiments of anti-CCR4 IgGs using labelled ligands (Example 3). Novalues were determined for KW0761. The quality of the fits is judged byleast-square (R2) values.

Table 21 is an overview of antagonistic properties of anti-CCR4antibodies in ligand mediated Ca-Flux experiments (Example 5). In caseof TARC-induced Ca-Flux, IC50 values were determined and quality of thefits is judged by least-square (R2). The inhibitory effect onMDC-induced signaling is presented as maximum inhibition of anti-CCR4antibodies at 10 μg/ml, expressed in %. No values were determined forKW0761.

Table 22 is an overview of determined IC50 values and remainingmigration in from inhibition of ligand-induced migration (TARC and MDC)by anti-CCR4 antibodies. IC50 values were derived from titration curvesin presence of ligands as described in Example 6. The quality of thefits is judged by least-square (R2). In case of inhibition of MDC,maximum inhibition at an IgG concentration of 10 μg/ml is presented.

Table 23 is an overview of determined IC50 values from inhibition ofTARC-induced invasion by anti-CCR4 antibody 503 on 786-0 cells. IC50values were derived from titration curves in presence of TARC (25 and125 nM) as described in Example 6. The quality of the fits is judged byleast-square (R2).

Table 24 is an overview of determined EC50 values from ADCC experimentsas described in Example 9 on haematological tumor cell lines CCRF-CEMand L-428. The determined EC50 values were derived by fitting data to a“log(agonist) vs response model” using GraphPad (Prism). Maximumcyotoxicity values are presented at the given concentration.

Table 25 Scoring table from IHC experiments for renal cell cancer tumormicroarrays (TMA) and relevant control tissues (placenta and normalkidney). Number of cores staining positive are given out of a totalnumber of cores stained on one TMA. The number of cores stained differsfor each staining since damaged cores were not scored. Zero refers to nostaining, 1 refers to intermediate staining and 2 refers to strongstaining.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 a) and 1 b) show the results of Example 2: Binding of anti-CCR4scFvs to target cells, expressing CCR4. ScFvs were cross-linked usinganti-Myc-antibody (mouse) to simulate a dimeric situation and diluted3-fold, starting at 10 μg/ml. Bound scFvs were detected usingRPE-conjugated anti-mouse IgG. FIG. 1 a) Binding to CCR4-positive DT40cells. FIG. 1 b) Binding to CCR4-negative DT40 cells.

FIG. 2. Results of Example 2: Binding of anti-CCR4 scFv 208 to Hek293Tcells, expressing CCR4 in comparison to a CCR4-negative Hek293T cells.ScFv was cross-linked using anti-Myc-antibody (mouse) to simulate adimeric situation and diluted 3-fold, starting at 10 μg/ml. Bound scFvswere detected using RPE-conjugated anti-mouse IgG.

FIG. 3. Results of Example 2: Binding of anti-CCR4 scFvs to naturaltarget cell (CCRF-CEM), expressing CCR4. ScFvs were cross-linked usinganti-Myc-antibody (mouse) to simulate a dimeric situation and diluted3-fold, starting at 20 μg/ml. Bound scFvs were detected usingRPE-conjugated anti-mouse IgG.

FIG. 4. Results of Example 3: Ligand-interfering binding experiments ofanti-CCR4 scFvs using Alexa647-labeled MDC-SNAP. ScFvs were incubated ata concentration of 5 μg/ml (150 nM) and diluted 3-fold over twelvedilution points in the presence of a fixed concentration of MDC-SNAP (50nM).

FIG. 5. Results of Example 3: Ligand-interfering binding experiments ofanti-CCR4 scFvs in presence of TARC and MDC. ScFvs were incubated at aconcentration of 0.5 μg/ml in presence of 2.5 μg/ml mouseanti-Myc-antibody (mouse) in presence of a fixed concentration ofligands (1 μg/ml) and stained using an anti-mouse-RPE conjugatedantibody. ScFvs as described in Example 1 (candidate 601 not presented).Signals were compared to cells, stained only with antibody-controls(background) as well as to unstained cells (unstained).

FIG. 6. Results of Example 4: Competition experiment between anti-CCR4antibodies 208, 306, 308, 406, 501, 503, 601, 60, 803 and KM3060var inpresence of biotinylated KW0761-IgG. The biotinylated KW0761-IgG wasdetected using PE-conjugated Streptavidin

FIG. 7. Results of Example 5: Inhibition of TARC-mediated Ca-Flux byanti-CCR4 scFv antibodies in comparison to KM3060var. TARC was incubatedat a final concentration of 28.6 ng/μl (3.6 nM) in presence ofincreasing scFv-concentrations, titrated over six dilution points,starting at 0.5 μg/ml. The signals were expressed in % where 100%signaling refers to recorded signals in presence of TARC-ligand, but noscFv antibody.

FIG. 8. Results of Example 5: Inhibition of MDC-mediated Ca-Flux byanti-CCR4 scFv antibodies. MDC was incubated at a final concentration of5 ng/μl (6.25 nM) in presence of a fixed scFv concentration of 10 μg/ml.The signals were expressed in % where 100% signaling refers to recordedsignals in presence of MDC-ligand, but no scFv antibody.

FIG. 9. Results of Example 6. Inhibition of TARC-mediated chemotaxis onCCR4+ CCRF-CEM cells in presence of increasing concentrations ofanti-CCR4 scFv antibodies (0.36 to 333 nM). TARC had a fixedconcentration of 3.5 nM. The signals were expressed in % where 100%refers to migration of cells in presence of TARC-ligand, but no scFvantibody. The graphs were fitted.

FIG. 10. Results of Example 2, binding of anti-CCR4 IgGs to target cellsexpressing CCR4. IgGs were incubated at defined concentrations and boundIgGs were detected using RPE-conjugated anti-Human IgG. a.) Binding toCCR4-positive CCRF-CEM cells. IgGs were diluted four-fold over eightpoints starting at 1 μg/ml. b.) Binding to CCR4-positive Hut78 cells.IgGs were diluted four-fold over eight points starting at 1 μg/ml. c.)Binding to CCR4-positive L-428 cells. IgGs were diluted three-fold overeight points starting at 10 μg/ml.

FIG. 11. Results of Example 2, binding to CCR4-expressing target cells(CCRF-CEM) in presence of human serum. Biotinylated samples wereincubated at 10 μg/ml in either 0% human serum (FACS-buffer) or in 10and 50% human serum. Samples were detected viaPE-conjugated-Streptavidin. a.) Comparison of recorded binding signals.b.) Remaining binding signals converted into %.

FIG. 12. Results of Example 3, Ligand-interfering binding experiments ofanti-CCR4 IgGs using labelled ligands. a.) IgGs were incubated at aconcentration of 5 μg/ml (35 nM) and diluted 4-fold over eight dilutionpoints in the presence of a fixed concentration of Alexa647 labeledMDC-SNAP (50 nM).b.) IgGs were incubated at a concentration of 2.5 μg/ml(17.5 nM) and diluted 4-fold over eight dilution points in the presenceof a fixed concentration of biotinylated TARO (3.12 μM). Remaining boundbiotinylated TARO was detected using PE-conjugated Streptavidin.

FIG. 13 Results of Example 6: Inhibition of CCL17/TARC-mediated invasionof 786-0 cells. Cells were incubated at two different concentrations ofCCL17/TARC (25 and 125 nM) in presence of increasing concentrations ofanti-CCR4 antibody 503 (μg/ml). Migrated cells were stained and analyzedas outlined in Example 6 and converted in %. The dotted line (y=25%)indicates the basal invasive potency of the 786-0 cells during theassay.

FIG. 14 Results of Example 9: Induction of ADCC on CCR4-expressing celllines CCRF-CEM (a) and L-428 (b). Calcein-labeled target cells wereincubated at different concentrations of anti-CCR4-antibodies asdescribed in Example 1 in presence of human PBMCs. KW0761 was includedas control. Induction of ADCC was converted into % based on fluorescenceintensity of the samples with 100% cell lysis after treatment withTritonX-100. The dose-response curves were computed by nonlinearregression analysis.

FIG. 15 Results of Example 9: Induction of ADCC on CCR4-expressingisolated Treg-cells. Treg cells were isolated as described in Example 9,calcein-labeled and incubated with a single concentration of anti-CCR4antibodies at 4.5 μg/ml in presence of autologous PBMCs. Cytotoxicitywas converted into % by normalizing to maximum release of calcein fromcells in presence of Triton. The percentage of killing is indicated.

FIG. 16 Results of Example 10: In vivo efficacy of anti-CCR4 antibodiesin a human xenograft model of adult T-cell lymphoma leukemia (ATLL).Presented are the measured body weights of the different groups duringthe study after tumor implantation (TI). Group A (control group) wassacrificed after 23 days due to large tumor volumes. a.) Mean bodyweight (in g) over time of treatment. b.) Relative body weight (in %)over time of treatment.

FIG. 17 Results of Example 10: In vivo efficacy of anti-CCR4 antibodiesin a human xenograft model of adult T-cell lymphoma leukemia (ATLL).Comparison of measured tumor volumes of the different groups (a-e)during the study after tumor implantation (TI). Individual tumor volumes(measured in mm3) are plotted against time post tumor implantation (TI).The control group (Group A) had to be sacrificed at day 23 due to largetumor volumes.

FIG. 18 Results of Example 10: In vivo efficacy of anti-CCR4 antibodiesin a human xenograft model of adult T-cell lymphoma leukemia (ATLL). a.)Tumor mean values between the different experimental groups. The numberof alive animals within each group is indicated. b.) Survivalproportions, calculated by comparison of individual treated group(antibody) vs untreated (control) group. Statistical significant valueswere identified for anti-CCR4 antibody 306 and 503. c.) Calculated tumordoubling times at day 23. Statistical significant difference wasidentified for anti-CCR4 antibody 503 (marked with an *).

FIG. 19 Results of Example 12: Binding of anti-CCR4 antibody 503 totumor microarray from renal cell cancer (RCC) patients. Binding of IgG503 was assessed on paraffin-embeded tissue sections. Antibody wasincubated at a concentration of 3 μg/ml and visualized upon incubationwith goat biotin-conjugated anti-human antibody (1:500) and visualizedusing the vectastain elite ABC kit with 3,3 diaminobenzidine assubstrate. The magnification of the microscope is given in x-fold. Thestaining was optimized on positive control tissue from spleen (a.) andconfirmed for specificity on normal human placenta tissue (b.). Bindingto tissue from RCC patients is presented in c.) and d.).

EXAMPLES Comparative Information

As discussed above, a known anti-CCR4 antibody family comprises KM2160,KM3060, KM2760 and KW-0761, which recognise an epitope existing in aregion of positions 2-29 from the N-terminal amino acid of human CCR4(EP1270595).

In some of the experiments reported herein, the present inventors haveused KM3060var (also referred to herein as “KM3060v”), which correspondsto KM3060, but potentially has a different sugar profile because it wasexpressed in a different host. This antibody was used in the scFvformat.

In some of the experiments reported herein, the present inventors haveused KW0761 in the IgG format.

KM2760 has been reported not to block the interaction between CCR4 andTARC or MDC (Ishida et al. 2006, Cancer Research 66 (11), pp 5716-5722).The applicant's findings using KM3060var are consistent with thisreport.

KM2760 has also been reported not to inhibit CCR4 signaling. Theapplicant has found that KM3060var does not inhibit MDC or TARC-inducedcalcium flux. The applicant has also found no significant inhibition ofMDC or TARC-mediated chemotaxis by KM3060var.

Example 1 Novel Antibodies

Nine human antibodies have been identified which can specifically bindto CCR4. Single chain forms of the antibodies were cloned in the pHOG21plasmid which contains a c-myc and 6xHis tag epitopes. TG1 bacteria weretransformed, and the scFv was expressed upon IPTG induction. The bindingof the purified scFv was confirmed by EasyCyte (see Example 2).

The nucleotide sequences of the heavy and light chain of the antibodyproducing clones were sequenced. The antibodies are designated as 208,306, 308, 406, 501, 503, 601, 603 and 803. The sequences of inter aliathe CDR regions of the light and heavy chains of 208, 306, 308, 406,501, 503, 601, 603 and 803 are shown in Tables 1 to 9 respectively.

The IgG form of antibodies 208, 306, 308, 406, 501, 503, 601, 603 and803 has also been made. IgGs were prepared using standard protocols.Briefly, the genes encoding the corresponding variable domains werecloned into the mammalian expression vector pLNO comprising the genesfor human constant domains (Norderhaug et al, 1997). The antibodies wereexpressed in a cell factory, and the first harvest was purified on aprotein A column and fractionated into monomer by size exclusionchromatography. The IgGs retained their ability to specifically bind toCCR4.

The IgG form of these antibodies is of the IgG1 isotype and it comprisestwo heavy chains and two light chains. Each heavy chain comprises a VHdomain (sequences shown in the relevant Tables), and a human IgG1constant region. Each light chain comprises a VL domain (sequences shownin the relevant Tables), and a human lambda light constant region. Asexplained in Example 7, defucosylated forms of the IgG antibodies weremade. Any assays described herein which use IgG form of the antibodiesof the invention use the defucolysated form.

Example 2 Binding of Anti-CCR4 Antibodies to Target Expressing Cells

To demonstrate the CCR4-specificity of the antibodies disclosed inExample 1, in-house CCR4 transfected and untransfected HEK293T-cells,DT40-cells and the natural CCR4+CCRF-CEM cell line were used in flowcytometry for staining with scFvs 208, 306, 308, 406, 501, 503, 601, 603and 803. As a positive control, in-house cloned and expressed KM3060varscFv was used. An anti-GFP scFv-antibody (raised against the greenfluorescent protein) was used as negative control.

CCRF-CEM (acute lymphoblastic leukemia, ATCC number CCL-119), HEK293T/17(human kidney, ATCC number CRL-11268), and DT40 (chicken lymphoma, ATCCnumber CRL-2111) cell lines were obtained from the American Type CultureCollection (ATCC, Rockville, Md.).

For transient transfection with human CCR4, Hek293T/17 cells were seededas 2×106 cells in a T75 (NUNC) flask. 48 h after seeding, the cells weretransfected with pcDNA3.1 plasmid encoding human CCR4 using Fugene(ROCHE) as transfecting agent. 40 μl Fugene and 16 μg DNA are used perT75. The cells were used for assays 48 h after transfection.

The CCRF-CEM and DT40 cells were maintained in RPMI-1640 culture mediumand the HEK293T cells were maintained in Dulbecco's Modified EagleMedium (DMEM) culture medium. All cells were maintained with fetal calfserum, the concentration was 10% for DT40 and HEK293T cells and 20% forCCRF-CEM cells. All media were supplemented with Penicillin andStreptomycin.

For the flow cytometry experiments, the cells were harvested from theculture flasks, washed 2 times with PBS, re-suspended in PBS with 0.2%BSA and 0.09% NaN3 and finally aliquoted 1×105 cells per well intoV-shaped 96-well plates (Greiner Bio-One, Frickenhausen, Germany). Cellswere centrifuged at 400×g for 5 min and then incubated at 4° C. for 60min with different scFv dilutions.

All scFv preparations were pre-incubated with a mouse anti-myc-antibody(9E10.2, Diatec, Oslo, Norway) to cross-link scFvs prior adding to thecells and diluted 3-fold, starting at a concentration of 10 μg/ml.

After washing with PBS with 0.2% BSA and 0.09% NaN3, the cells werestained with 1 μg/ml of RPE-conjugated goat anti-mouse IgG (AbD Serotec,Duesseldorf, Germany) for 30 minutes at 4° C. The stained cells werewashed, re-suspended in 200 μl PBS with 0.2% BSA and 0.09% NaN3 andtransferred to a U-shaped 96-well plate (Corning, Schiphol-Rijk, TheNetherlands) for acquisition on EasyCyte flow cytometer (GuavaTechnologies, Hayward, Calif., USA).

The results obtained clearly indicate that the scFvs 208, 306, 308, 406,501, 503, 601, 603 and 803 are specific for CCR4. Binding of scFvs 208,306, 308, 406, 501, 503, 601, 603 and 803 to CCR4-positive DT40 cellscompared to CCR4-negative DT40 cells is illustrated in FIGS. 1 a) and 1b). Selective binding of scFvs 208, 306, 308, 406, 501, 503, 601, 603and 803 to CCR4+HEK293T cells compared to CCR− HEK293T cells was alsoshown (FIG. 2; data only shown for antibody 208). Binding of scFvs 208,306, 308, 406, 501, 503, 601, 603 and 803 to CCR4-expressing CCRF-CEMcells is illustrated in FIG. 3.

CCR4 specificity was also confirmed for antibodies 306, 406 and 503 inthe defucosylated IgG1 format (see Example 7) by comparing binding toHEK293T/17 cells and HEK293T/17 transformed to express human CCR4. Theseantibodies were also tested for binding to the CCR4-positive cell linesCCRF-CEM, L-428, Hut78, 786-0, A498, KatoIII and MCF-7 using flowcytometry.

As positive controls, either anti-CCR4 antibody 1G1 (BD Pharmingen,Franklin Lakes, N.J., USA.), anti-CCR4 antibody Ab1669 (Abcam,Cambridge, UK) as well as in house produced KW0761 IgG (see above) wereused. Isotype control antibodies 6-MAM which binds the opioid drugheroin and an anti-GFP-antibody (raised against the green fluorescentprotein) were used as negative controls. Both antibodies were producedin house as IgG1-molecules.

To demonstrate binding to target expressing cells in presence of humanserum, anti-CCR4 antibodies 306, 406, 503 and KW0761 were biotinylatedvia Cysteines following the manual of the EZ-link maleimide-PEG solidphase biotinylation kit (Thermo Fisher, Rockford, Ill. USA).

The CCR4-positive cell lines CCRF-CEM, Hut78 (cutaneous T-cell lymphoma,ATCC number TIB-161), 786-0 (human renal cell carcinoma, ATCC numberCRL-1932), MCF-7 (human breast adenocarcinoma, ATCC number HTB-22),KatoIII (human gastric cancer, ATCC number HTB-103) and HEK293T/17 celllines were obtained from the American Type Culture Collection (ATCC,Rockville, Md., USA). L-428 cells (Non-Hodgkin Lymphoma, DSMZ number ACC197) and A-498 (human renal cell carcinoma, DSMZ number ACC 55) wereobtained from the “Deutsche Sammlung von Mikroorganismen andZellkulturen” (DSMZ, Braunschweig, Germany). The CCRF-CEM, L-428, Hut78,786-0 and KatoIII cells were maintained in RPMI-1640 culture medium.HEK293T/17 and MCF-7 cells were maintained in Dulbecco's Modified EagleMedium (DMEM; from PAA, cat# E15-810)) culture medium. A-498 cells werecultured in EMEM-medium. All cells were maintained with 10% of fetalcalf serum (from PAA cat# A15-252), except for CCRF-CEM, L-428, Hut78and KatoIII which were maintained in presence of 20% fetal calf serum.All media were supplemented with Penicillin and Streptomycin (from PAAcat#P11-010).

For the flow cytometry experiments, the suspension cell lines CCRF-CEM,Hut78 and L-428 were harvested directly from the culture flasks.Adherent cell lines Hek293T/17, 786-0, A-498, KatoIII and MCF-7 werewashed 2 times with PBS and detached from the culture flasks byincubating with Accutase (PAA laboratories, Linz, Austria) for 3 minutesat room temperature according to the manufacturer's protocol. All cellswere re-suspended in PBS with 0.2% BSA and 0.09% NaN3 and finallyaliquoted 1×105 cells per well into V-shaped 96-well plates (GreinerBio-One, Frickenhausen, Germany). Cells were centrifuged at 400×g for 5min and then incubated at 4° C. for 60 min with different IgG dilutions.For binding in presence of human serum, CCRF-CEM cells were incubatedwith different IgG-solutions at either 10% or 50% of human serum (SigmaAldrich, St Louis, Mo., USA) at 37° C. for 60 min.

After washing with PBS with 0.2% BSA and 0.09% NaN3, the cells werestained with 1 μg/ml of RPE-conjugated goat anti-Human IgG (AbD Serotec,Duesseldorf, Germany) for 30 minutes at 4° C. The stained cells werewashed, re-suspended in 200 μl PBS with 0.2% BSA and 0.09% NaN3 andtransferred to a U-shaped 96-well plate (Corning, Schiphol-Rijk, TheNetherlands) for acquisition on EasyCyte (Guava Technologies, Hayward,Calif., USA).

The results obtained clearly confirm that the IgG antibodies 306, 406and 503 are specific for CCR4. Binding of IgGs 306, 406 and 503 toCCR4-positive Hek293T/17 cells was significantly higher than binding toCCR4-negative Hek293T/17 cells. Control antibody 1G1 also bound toCCR4-positive Hek293T/17 cells, but gave a weaker signal.

Binding of IgG antibodies 306, 406 and 503 to haematologicalCCR4-expressing L428 cells and Hut78 cells, as well as a CCRF-CEM cells,is illustrated in FIG. 10. Binding of 306, 406 and 503 to 786-0, A498,KatoIII and MCF-7 was also confirmed, whereas the negative controlantibodies did not bind to these cell lines (data not shown).

Reassuringly, binding of the anti-CCR4-antibodies to CCR4-positive cellsis not inhibited in presence of increasing concentrations of humanserum, as illustrated in FIG. 11 in relation to the cell line CCRF-CEM.

FIG. 11 also shows that binding of antibodies 306, 406 and 503 issuperior to binding of the antibody KW0761. Similar observations weremade when biding to the cell lines KatoIII, MCF-7 and A498 was assayed(data not shown).

The binding profile of the anti-CCR4-antibodies 306, 406 and 503 to thedifferent CCR4+ cell lines shows increased binding to some of the CCR4+cell lines compared to other CCR4+ cell lines. For instance, binding toL-428 is decreased in comparison to CCRF-CEM. L-428 are known to secretethe CCR4-ligand TARC (see Ishida T et al, Leukemia Vol 20, 2006) and theanti-CCR4 antibodies 306, 406 and 503 compete for the CCR4-binding sitewith TARC (see Example 3).

The binding profile of the anti-CCR4-antibodies 306, 406 and 503 differsfrom that of KW0761. For example, KW0761 shows increased binding toL-428. Without wishing to be bound by theory, this is believed to be dueto different epitope binding sites of these antibodies (as outlined inExample 4). KW0761 does not block the binding of CCR4 and TARC, so itdoes not compete with the TARC secreted by L-428.

In addition, EC50 values were determined (data not shown) with varyingvalues from cell line to cell line, which is believed to be due todifferences in CCR4 expression on the surface of the various cell types.

Example 3 Anti-CCR4 Antibodies Interference with Ligand Binding

To determine whether the anti-CCR4 antibodies from Example 1 interferewith the binding of CCR4 ligands to the receptor, competitionexperiments were performed. To this end, CCR4-positive CCRF-CEM targetcells were incubated at a fixed concentration of MDC-SNAP in presence ofincreasing concentrations of scFvs. MDC was genetically fused to theN-Terminus of the SNAP-tag such that the SNAP tag is fused to theC-terminus of MDC. The SNAP-tag is derived from the 20 kDa DNA repairprotein 06-alkylguanine-DNA alkyltransferase. The gene was ordered atGeneart (Regensburg, Germany) and HEK293/T cells were transientlytransfected with the gene, encoding for the MDC-SNAP-fusion protein.After 5-6 days, the fusion-protein was purified via a Ni-NTA-affinitycolumn followed by size exclusion to isolate the monomeric MDC-SNAPfraction. MDC-SNAP was labeled with Alexa647, following the proceduresin the manual of the Alexa Fluor 647 Molecular Probe labelling kit(Invitrogen Corporation, San Diego, Calif.). The functionality of theMDC-SNAP to bind CCR4 was confirmed in a chemotactic assay on CCRF-CEMcells (data not shown).

The CCR4+ CCRF-CEM-cells were harvested from culture flasks, washed 2times with RPMI-1640 culture medium and aliquot 1×105 cells per wellinto V-shaped 96-well plates (Greiner Bio-One, Frickenhausen, Germany).Cells were centrifuged at 500×g for 5 minutes and the supernatant wasaspirated. ScFvs 208, 306, 406, 501, 503, 601, 603 and 803 (sample 308was not included) were diluted 3-fold starting at 5 μg/ml (150 nM) overeight dilution points and incubated for 60 minutes at 4° C. in presenceof a fixed concentration of 140 ng/ml of MDC-SNAP (50 nM) in PBScontaining 0.2% BSA and 0.09% NaN3 on the cells. After washing threetimes with PBS containing 0.2% BSA and 0.09% NaN3, the cells werere-suspended in 200 μl PBS with 0.2% BSA and 0.09% NaN3 and transferredto a U-shaped 96-well plate (Corning, Schiphol-Rijk, The Netherlands)for flow cytometry using a FACS Canto II flow cytometer (BD Biosciences,Heidelberg, Germany) to record the signal of the Alexa647 labeledMDC-SNAP fusion protein. The results showed a clear decrease in stainingsignals with increasing scFv concentrations (FIG. 4). This indicatesthat the antibodies interfere with the ligand binding and thus have aCCR4-blocking activity.

A similar experiment was performed to test whether the scFvs describedin Example 1 interfere with TARC- and MDC-ligand binding to CCR4.Therefore, the CCR4+DT40-cells were harvested from culture flasks,washed 2 times with RPMI-1640 culture medium and aliquoted 1×105 cellsper well into V-shaped 96-well plates (Greiner Bio-One, Frickenhausen,Germany). Cells were centrifuged at 500×g for 5 minutes and thesupernatant was aspirated and then incubated for 30 minutes at 37° C.with 0 or 1 μg/ml of TARC or MDC (PeproTech EC, London, UK) in RPMI-1640culture medium. The supernatants were aspirated after a centrifugationstep at 500×g for 5 minutes and cells were incubated for 1 hour at 4° C.with 0.5 μg/ml of scFvs 208, 306, 308, 406, 501, 503, 601, 603 and 803and 2.5 μg/ml of a mouse anti-cMyc (9E10.2; DIATEC Monoclonals, Oslo,Norway). Cells were centrifuged at 500×g for 5 minutes and thenincubated for 45 minutes at 4° C. with 1 μg/ml of RPE-conjugated goatanti-mouse IgG (BD Pharmingen, San Diego, USA, CA). Cells were washed,re-suspended in 200 μl PBS with 0.2% BSA and 0.09% NaN3 and transferredto a U-shaped 96-well plate (Corning, Schiphol-Rijk, The Netherlands)for flow cytometry using an EasyCyte device (Guava Technologies,Hayward, Calif., USA). The results showed a clear decrease in stainingsignals when scFvs were incubated in presence of TARC and MDC (FIG. 5).This indicates that the scFvs described in Example 1 interfere with theligand binding and thus have a CCR4-blocking activity.

Substantially similar protocols were used to test the effect of the IgG1forms of 306, 406 and 503 on ligand binding. KW0761 was used forcomparison. The experimental data were fitted by non-linear regressioncurve fit using a model “log (inhibitor) vs. response” of software Prism(GraphPad) and are summarized in Table 20.

The results showed a clear decrease in staining signals of both labelledligands MDC and TARC with increasing concentrations of IgGs, except forKW0761 (FIG. 12). This indicates that the IgG1 forms of 306, 406 and 503interfere with the ligand binding and thus have a CCR4-blockingactivity.

Example 4 Competition Between Anti-CCR4 Antibodies

To analyze the epitope recognized by the anti-CCR4 antibodies 208, 306,308, 406, 501, 503, 601, 603 and 803 in comparison to KM3060var,competition experiments were performed using flow cytometry. The bindingof biotinylated KW0761 IgG antibody to CCR4+CCRF-CEM cells waschallenged in the presence of different concentrations ofnon-biotinylated anti-CCR4 antibodies (208, 306, 308, 406, 501, 503,601, 603, 803 and KM3060var) in single chain format TheCCR4+CCRF-CEM-cells were harvested from culture flasks, washed 2 timeswith RPMI-1640 culture medium and aliquot 1×105 cells per well intoV-shaped 96-well plates (Greiner Bio-One, Frickenhausen, Germany). Cellswere centrifuged at 500×g for 5 minutes and the supernatant wasaspirated. Antibody KW0761-IgG was biotinylated using standard methods.The biotinylated KW0761-IgG was incubated on CCRF-CEM cells for 1 hourat 4° C. at a fixed concentration of 1 μg/ml in presence of anti-CCR4scFvs 208, 306, 308, 406, 501, 503, 601, 603, 803 and KM3060var whichwere three-fold diluted over eight titration points, starting at 5μg/ml. After washing with PBS with 0.2% BSA and 0.09% NaN3, the cellswere stained with 2.5 μg/ml of Streptavidin-RPE (BD Pharmingen, SanDiego, Calif., USA) for 30 minutes at 4° C. for detection ofbiotinylated KW0761-IgG. The stained cells were washed, re-suspended in250 μl PBS with 0.2% BSA and 0.09% NaN3 and transferred to a U-shaped96-well plate (Corning,

Schiphol-Rijk, The Netherlands) for acquisition using an FACS Canto IIflow cytometer (BD Biosciences, Heidelberg, Germany).

The results shown in FIG. 6 illustrate that none of the anti-CCR4antibodies described in Example 1 compete with KW0761 for binding to thecells and only a competition between antibodies KW0761-IgG and KM3060varscFv is observed. This indicates that the antibodies 208, 306, 308, 406,501, 503, 601, 603 and 803 do not bind to the same epitope as KM3060varor KW0761.

A substantially similar protocol was used to assay competition of theIgG1 forms of 306, 406 and 503 with KW0761 and with one another. Thisexperiment confirmed that antibodies 306, 406 and 503 compete with oneanother, but not with KW0761 (data not shown).

Example 5 Antagonistic Activity

The ability of the antibodies 208, 306, 308, 406, 501, 503, 601, 603 and803 to reduce ligand induced calcium flux in the natural CCR4+ CCRF-CEMcell line was assessed. The CCRF-CEM target cells, cultivated underregular conditions, were sedimented by centrifugation and resuspendedtwice in RPMI-1640 culture medium. One ml with 2.5×106 cells was mixedwith Fluo-4-AM (Invitrogen, Carlsbad, Calif.), Pluronic F-127(Invitrogen, Carlsbad, Calif.) and Probenecid to final concentrations of1 μM, 0.02% and 1 mM respectively. The cells were incubated at 37° C.for 30 min on a vertical rotating wheel (7 rpm). All subsequent stepswere done in the presence of 1 mM Probenecid. The cells were washedtwice in RPMI-1640 with 10% FCS, once in assay buffer (145 mM NaCl, 4 mMKCl, 1 mM NaH2PO4, 0.8 mM MgCl2, 25 mM Hepes, 22 mM glucose). Theinhibition of TARC and MDC-mediated Ca-Flux was divided into two assays.

The inhibition of TARC-mediated Ca-Flux in presence of scFvs 208, 306,308, 406, 501, 503, 601, 603 and 803 was evaluated using the PheraStarFShigh-through put reader (BMG Labtech, Offenburg, Germany). The antibodyKM3060var was used as a negative control. The cells were diluted to afinal concentration of 1.6×106 cells/ml. A volume of 25 μl of cells wastransferred to a black 96-well plate (Nunc, Thermo Fisher Scientific,Rochester, N.Y., USA) and incubated for 15 min at room temperature inpresence of 25 μl of scFv antibodies in the dark. ScFvs were titratedten-fold over six titration points, starting at 0.5 μg/ml. All scFvswere applied in triplicates, sample KM3060var was included forcomparison. The plate was transferred to the PheraStarFS reader and theligand was automatically injected to each well separately at a finalconcentration of 28.6 ng/μl (3.6 nM). The changes in fluorescence weremeasured over a total of 16 points using the 485-520 nm band pass filter(5 points before ligand injection (range 1); 1 point during the ligandinjection (start of range 2); 10 points after the injection of theligand (range 2)). The % of activation was calculated using followingequation:

$\frac{{\begin{bmatrix}{{Sum}\mspace{14mu} {of}\mspace{14mu} {range}} \\{{2\mspace{14mu} {raw}\mspace{14mu} {data}} -} \\\left( {11 \times {start}\mspace{14mu} {of}\mspace{14mu} {range}\mspace{14mu} 2} \right)\end{bmatrix}{IgG}} - {\begin{bmatrix}{{Sum}\mspace{14mu} {of}\mspace{14mu} {range}} \\{\; {{2\mspace{14mu} {raw}\mspace{14mu} {data}} -}} \\\left( {11 \times {start}\mspace{14mu} {of}\mspace{14mu} {range}\mspace{14mu} 2} \right)\end{bmatrix}{buffer}}}{\begin{bmatrix}{{{Sum}\mspace{14mu} {of}\mspace{14mu} {range}\mspace{14mu} 2\mspace{14mu} {raw}\mspace{14mu} {data}} -} \\\left( {11 \times {start}\mspace{14mu} {of}\mspace{14mu} {range}\mspace{14mu} 2} \right)\end{bmatrix}{Ligand}}$

The inhibition of MDC-mediated Ca-Flux in presence of scFvs 208, 306,308, 406, 501, 503, 601, 603 and 803 was evaluated using a FACSCanto IIflow cytometer (BD Biosciences, Heidelberg, Germany). Cells were stainedas described above and diluted to a final concentration of 1.2×106cells/ml. Equal volumes of cells, assay buffer with or without scFvantibodies and ligand (MDC) were mixed. The first two components (cellsand antibodies) were pre-incubated for 15 min prior to addition of theligand. The final concentrations of the scFvs were 10 μg/ml whereas MDCwas 5 ng/ml. The samples were immediately analyzed using the 515-545 nmband pass filter on a FACSCanto 11 flow cytometer (BD Biosciences,Heidelberg, Germany). For evaluation, the average signals of stainedcells, but otherwise untreated, was subtracted from all samples. Thesignal recorded from stained cells in presence of MDC, but no scFvantibody, was set as basis for 100% signaling and cells in presence ofMDC and scFv antibodies were converted into %.

The results shown in FIGS. 7 and 8 clearly demonstrated that all scFvsdescribed in Example 1 act as inhibitors of TARC and MDC. The IC50values derived from the TARC inhibition are presented in Table 19.

A substantially similar protocol was used to assay the effect onligand-induced calcium flux by the IgG1 forms of 306, 406 and 503compared with KW0761. This experiment confirmed that antibodies 306, 406and 503, but not KW0761, reduce ligand-induced calcium flux. Theexperimental data were fitted by non-linear regression curve fit using amodel “log (inhibitor) vs. response” of software Prism (GraphPad).

The results clearly demonstrate that the antibodies of the invention actas inhibitors of TARC and MDC induced Ca-Flux. The IC50 values derivedfrom the TARC inhibition are presented in Table 21. It should beunderstood that a comparison of these deduced values with values deducedunder different experimental conditions would not necessarily beappropriate.

Example 6 Inhibition of Chemotaxis

The inhibition of chemotaxis by the scFv antibodies in Example 1 wasassayed by contacting CCR4+cells capable of chemotaxis with scFvantibodies in one chamber, whereas a ligand of CCR4 (MDC or TARC) wasplaced in another chamber separated from the first chamber by a membraneor filter having a suitable pore size. The effect of the antibodies oncell migration towards the ligand (chemotaxis) was determined bycomparing chemotaxis in the presence of the antibody to chemotaxis inthe absence of the antibodies. All scFv antibodies as described inExample 1 were found to inhibit chemotaxis of CCR4-positive cellstowards MDC and TARC.

CCR4+CCRF-CEM target cells, cultivated as described in Example 2, weresedimented by centrifugation and re-suspended twice in RPMI-1640 culturemedium without FCS. In a third centrifugation step, cells werere-suspended in RPMI-1640 culture medium supplemented with 1% FCS andadjusted to a final density of 1.6×107 cells/ml. In parallel, 150 μl ofRPMI supplemented with 1% FCS containing TARC or MDC at 3.5 nM was addedinto each of the lower chamber of a Multiscreen-MIC plate 96 well (8 μmpores, Millipore, Billerica, Mass., USA, MAMIC5S10). ScFv antibodieswere serial diluted from 333 nM down to 0.36 nM in presence of a mouseanti-myc-antibody (9E10.2, Diatec, Oslo, Norway) to cross-link scFvs andsimulate a dimeric IgG format. 50 μl of the scFv antibodies were mixedwith 50 μl of cells (final concentration of 3.9×105 cells/ml) and addedto the upper compartment of the Multiscreen-MIC 96 chamber plate andincubated at 37° C. for 3 hr. The filter was removed and 100 μl of cells(from the lower chamber) were transferred after re-suspending into a96-well COASTAR plate (Greiner Bio-One, Frikenhausen, Germany); anadditional volume of 100 μl of PBS (supplemented with 0.2% BSA and 0.09%NaN3) was added to the samples. Migration was evaluated by counting ofgated cells using a FACSCanto II flow cytometer (BD Biosciences,Heidelberg, Germany).

The number of migrated cells in presence of TARC or MDC, but no scFvantibody, was set as basis for 100% migration and the number of migratedcells in presence of ligands and scFv antibodies were converted into %.

The data presented in FIG. 9 demonstrate that all scFv antibodiesdescribed in Example 1 inhibited TARC induced chemotaxis of CCRF-CEMcells. In addition, the same scFv antibodies inhibited MDC-mediatedchemotaxis (data not shown).

A substantially similar experiment was carried out with the IgG1 formsof 306, 406 and 503, confirming that the IgG1 forms also inhibited TARCinduced chemotaxis of CCRF-CEM cells, as well as MDC induced chemotaxisof CCRF-CEM cells. The results were analyzed by fitting the data using alog (inhibitor) vs. response function using GraphPad Prism to extractIC50 values. Determined IC50 values are summarized in Table 22.

In a further experiment, the effect of anti-CCR4 antibody 503 (IgG1form) on the migration of the solid tumor cell line 786-0 in response toTARC was assayed using a transwell plate assay.

A multiscreen-MIC 96 well plate (8 μm pores, Millipore, Billerica,Mass., USA, MAMIC8S10) was prepared by aliquoting 50 μl of a 1 μg/ml ofa matrigel solution (BD Matrigel, Cat. No. 356230, BD Biosciences,Heidelberg, Germany) on top of the pores for coating. The matrigel wasallowed to polymerize for 1 h at 37° C. Non-reacted matrigel was removedby washing the pores with PBS. The 786-0 cell were cultivated asoutlined above. Cells were harvested from flasks after washing 2 timeswith PBS and detached from the culture flasks by incubating withAccutase (PAA laboratories, Linz, Austria) for 3 minutes at roomtemperature. Cells were aliquoted to a final cell density of 8.0×105cells/ml in RPMI-1640, supplemented with 0.1% fetal calf serum. Inparallel, IgG antibody 503 was diluted to a concentration of 60 μg/mland serial diluted five-fold over 5 points in RPMI-1640 supplementedwith 0.1% FCS. The amount of salt present in the formulation buffer ofthe antibody was kept constant. The ligand TARC was diluted to two finalconcentrations, 1000 ng/ml and 200 ng/ml in RPMI-1640 supplemented with0.1% FCS. The amount of salt present in the formulation buffer of theantibody was kept constant. A volume of 150 μl of the ligand wasaliquoted in the bottom chamber of a transwell Multiscreen-MIC plate 96well plate (8 μm pores, Millipore, Billerica, Mass., USA, MAMIC8S10). Anequal volume of 50 μl of cells were mixed with a volume of 50 μl of theantibody dilutions. The mixture was placed on top of matrigel coatedmembrane and incubated for 3 h at 37° C. Not-migrated cells on top ofthe matrigel coated membrane were mechanically removed by scraping,whereas cells migrated through the matrigel coated membrane were stainedaccording to the manual of the cell titer glo luminescent cell viabilityassay kit (Promega, Madison, Wis., USA). The stained samples weretransferred to a 96-well plate (NUNC, flat bottom, black) and theluminescence was analyzed on a Tecan Infinite M200 reader (Tecan,Maennerdorf, Switzerland). The number of migrated cells in presence ofTARC, but without antibody, was set as basis for 100% migration and thenumber of migrated cells in presence of ligand and antibody wereconverted into %. The results were analyzed by fitting the data using alog (inhibitor) vs. response function using GraphPad Prism to extractIC50 values. The results show that antibody 503 inhibits TARC-inducedmigration of the solid tumor cell line 786-0 (FIG. 13). Determined IC50values are summarized in Table 23.

Similar findings were made on the murine renal cell cancer cell lineRENCA where anti-CCR4-antibody 503 was able to block murine TARC inducedmigration of the target cells (data not shown).

Example 7 Generation of Defucoylated Anti-CCR4 IgG1 Molecules

It has been reported that ADCC enhancement can be achieved bymanipulating the state of oligosaccharides on human IgG1 subclass (NiwaR et al, CanRes, Vol. 64, 2004). Modifying the amount of fucose wasdemonstrated to have a beneficial effect on ADCC. Therefore, antibodies306, 406 and 503 were produced in the presence of Kifunensine, aselective inhibitor of class I α-mannosidases, causing a stop infucosylation of the IgG during production in cell culture. AntibodyKW0761 was produced under the same conditions.

IgGs were produced in the presence of Kifunensine (100 ng/ml; SigmaAldrich, St Louis, Mo., USA). After harvesting the cell medium, IgGswere isolated first upon affinity purification using a ProteinA column(HiTrap, 5 ml, ProteinA; GE). IgGs were eluted using citrate buffer (pH3) and transferred into 1M Tris-buffer (pH 9). Prior a secondpurification, IgG samples were up-concentrated and loaded on to a sizeexclusion chromatography (HiLoad Sephadex 200, GE; running buffer 20 mMNa-Phosphate/145 mM NaCl, pH7.2). Monomeric fractions were collected andIgGs were up-concentrated a second time.

Example 8 Species Cross-Reactivity

Antibodies 306, 406 and 503 were tested for their ability to cross-reactwith CCR4 from species other than human.

For transient transfection, Hek293T/17 cells were seeded as 2×106 cellsin a T75 (NUNC) flask. 48 h after seeding, the cells were transfectedwith Fugene (ROCHE). 40 μl Fugene and 16 μg DNA are used per T75. Thecells were used for assays 48 h after transfection.

HEK-293T/17 cells were transiently transfected either with pcDNA3.1plasmid encoding human CCR4 or with pLNO plasmids (Norderhaug et al,1997) encoding either mouse CCR4 (GeneBank CAA62372) or monkey (Macacamulatta) CCR4 (PubMed access number XP_(—)001098807) using Fugene(Roche) as a transfection reagent. Non-transfected (CCR4 negative) cellsserved as a negative control. The cells were cultivated further for 48hours under regular conditions and harvested form flasks as describedabove. One times 105 cells per well were aliquoted into V-shaped 96-wellplates (Greiner Bio-One, Frikenhausen, Germany). Cells were centrifuged(400×g, 5 min, 4° C.), re-suspended in 50 μl of antibodies (306, 406,503 and isotype control 6-MAM) at a concentration of 1 μg/ml in PBScontaining 0.2% BSA and 0.09 NaN3 and incubated at 4° C. for 60 min.Expression of human and murine CCR4 was confirmed by incubatingaccording cells in presence of either PE-conjugated mouseanti-Human-CCR4 antibody 1G1 or with PE-conjugated hamsteranti-Mouse-CCR4 antibody 2G12 (Biolgend; San Diego, Calif., USA). Thesamples were then washed twice by centrifugation and re-suspension in150 μl FACS buffer. The cell pellets were finally re-suspended in 50 μlwith 3 μg/ml goat anti-human-IgG-PE (AbDSerotec, Dusseldorf, Germany)for detection of antibodies 306, 406, 503 and isotype control IgG 6-MAMand incubated at 4° C. for 45 min. The samples were washed twice asdescribed above and re-suspended in 250 μl FACS buffer followed bytransfer into a U-shaped 96-well plate (Corning, Schiphol-Rijk,Netherlands) for flow cytometry on FACSCantoll (BD Biosciences, SanJose, Calif.).

Antibodies 306, 406 and 503 were found to bind to both human and monkeyCCR4 and a reduced affinity to murine CCR4 was detected (data notshown).

Example 9 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

The ability of the anti-CCR4 antibodies 306, 406 and 503 to induce ADCCwas assessed and compared to KW0761 on the natural CCR4+ cell linesCCRF-CEM, L-428 and Hut78. In addition, induction of ADCC was tested onisolated regulatory T-cells (Treg-cells) using anti-CCR4 antibodies 306,406 and 503 in comparison to KW0761. It is well established thatTreg-cells play a key role in the immune-escape of solid tumor duringthe cancer disease progression (see Wolf A M et al, Clin Cancer Res Vol9, 2003). Importantly, CCR4 has been described to be expressed onCD4+CD25+ T cells which home to the stromal region of the tumor wherethey inhibit the immune response towards the tumor. Either inhibition ofmigration (induced by MDC) or direct killing (via ADCC) of theseTreg-cells via CCR4 could be a promising way to block metastasis or toreduce the growth of solid tumors.

For ADCC on the suspension cells CCRF-CEM, L-428 and Hut78, cells werecultivated under regular conditions as described in Example 2. Cellswere sedimented by centrifugation and re-suspended in RPMI-1640 culturemedium. This step was repeated once. One ml with 2.5×106 cells was mixedwith calcein-AM (Invitrogen, Carlsbad, Calif.) to a final concentrationof 10 pM and then incubated at 37° C. for 30 min on a vertical rotatingwheel (7 rpm). The cells were washed three times in RPMI-1640 with 10%FCS and the cell density was adjusted to 3×105/ml. Separately,peripheral blood mononuclear cells (PBMC) were prepared followingconventional procedures (enriched by Ficoll-Hypaque gradientcentrifugation), washed in RPMI-1640 with 10% FCS and re-suspended at6×106 per milliliter. Fifty μl of each target and effector cells wereadded to the same wells in a 96-well microtiter plate giving a ratio ofeffector to target cells (E:T) of 20:1. The antibodies were added inquadruplicate samples to the same wells in a volume of 100 μl resultingin a concentration range as follows: 0.2 μg/ml down to 0.002 ng/ml incase of CCRF-CEM; 1 μg/ml down to 0.32 ng/ml in case of L-428; 1 μg/mldown to 0.1 ng/ml in case of Hut78. The microtiter plate was thenincubated for four hours at 37° C., and 20 μl 0.9% Triton-X100 was addedto some of the wells after 3 hrs and 45 minutes to achieve completelysis of the target cells. One hundred μl supernatant of each sample wasthen transferred to a black microtiter plate and the fluorescence(excitation: 488 nm, emission: 518 nm) was analyzed in a TECAN InfiniteM200 plate reader (Tecan, Maennerdorf, Switzerland). The fluorescenceintensity in the samples with no antibodies was subtracted from theintensity of all other samples. The percentage of lysis in samples withantibodies was estimated based on fluorescence intensity of the sampleswith 100% cell lysis after treatment with TritonX-100. The dose-responsecurves were computed by nonlinear regression analysis and athree-parameter fit model using software Prism (GraphPad, San Diego,Calif., USA).

To test induction of ADCC on Treg-cells, the following protocol wasestablished and the setup was established using the anti-CCR4 antibody(1G1, Cat. No. #551266, BD Biosciences, Heidelberg, Germany).

The percentage of Treg cells in healthy donor blood is reported to bebetween 5 and 10% (see Wolf A M et al, Clin Cancer Res Vol 9, 2003).This is why the Treg cells had to be enriched from healthy donor PBMCsin order to get a decent amount of isolated Treg-cells. Isolation wasperformed according to the protocol from Dynabeads Regulatory CD4+CD25+T Cell Kit (Cat. No. #113-63D, Invitrogen Corporation, San Diego,Calif., USA), resulting in three different populations (PBMC, CD4+ andT-reg). At particular stages of the purification some cells were keptand appropriately stained as quality control. The PBMCs, the CD4+ andthe Treg cells were sedimented by centrifugation and re-suspended to1×106 cells/ml in PBS, supplemented with 0.2% BSA and 0.09% NaN3. Foreach experiment two hundred μl of non-separated 2×105 PBMCs and isolatedCD4+ cells and 100 μl with 1×105 isolated Treg were transferred in a 96well v-shaped plate and spun down at 400×g for 5 min at 4° C. Thesupernatants were discarded using a pipette, the cell pellets werere-suspended in either 50 μl of PBS supplemented with 0.2% BSA and 0.09%NaN3, in 50 μl of biotinylated anti-CD127 (Cat. No. #558633BDBiosciences, Heidelberg, Germany), which served for identification ofTreg cells (see Simonetta F et al, Eur J Immunol Vol 9, 2010) or 50 μlof biotinylated anti-CCR4 antibody (1G1, Cat. No. #551266, BDBiosciences, Heidelberg, Germany) and incubated at 4° C. for 1 h. Thesamples were washed 3 times with PBS (supplemented with 0.2% BSA and0.09% NaN3), spun down at 400×g for 5 min at 4° C. The supernatants werediscarded using a pipette, the cell pellets were re-suspended in either50 μl of PBS (supplemented with 0.2% BSA and 0.09% NaN3) or in a mixtureof 50 μl of anti-CD4-FITC and anti-CD25-APC (Cat. No. #11-0049-42 and#17-0259-42 eBiosciences San Diego, Calif., USA) and Streptavidin-PerCP(Cat. No. #554064, BD Biosciences, Heidelberg, Germany) with or withoutbiotinylated anti-CCR4 (1G1, Cat. No. #551266, BD Biosciences,Heidelberg, Germany) and incubated at 4° C. for 1 h. The samples werewashed 3 times with PBS (supplemented with 0.2% BSA and 0.09% NaN3),spun down at 400×g for 5 min at 4° C. The supernatants were discardedusing a pipette, the cell pellets with anti-CCR4-PE and unstained werere-suspended either 200 μl of PBS (supplemented with 0.2% BSA and 0.09%NaN3) and transferred to a 96 well u-shaped plate. Compensation usingthe different fluorochromes was performed on the FACSCanto II (BDBiosciences, Heidelberg, Germany) and the samples were analyzed. Theremaining samples were stained with anti-FoxP3-antibody (Cat. No.#560046, BD Biosciences, Heidelberg, Germany) according to the protocoldescribed in the FoxP3 staining buffer kit (Cat. No. #00-5523-00,eBiosciences San Diego, Calif., USA). Cells were re-suspended in 200 μlof PBS (supplemented with 0.2% BSA and 0.09% NaN3) and transferred to a96 well u-shaped plate for analysis in flow cytometry using theFACSCantoll (BD Biosciences, Heidelberg, Germany). Isolated Treg cellswere judged as pure Treg cells by double positive signals onanti-CD4-FITC/anti-CD25-APC as well as by double positive signals onanti-FoxP3-PE/anti-CCR4 (detection of biotinylated 1G1 usingStreptavidin-PerCp). It was found that approximately 95% of isolatedcells were CD4+CD25+ cells and 66% of these were FoxP3-CCR4-positive.

The ADCC experiment to evaluate the effect of the anti CCR4 antibodiesdescribed in example 1 on isolated Treg cells was performed asdescribed, except that the E:T (effector cells in this case isolatedfrom autologous PBMCs; target cells=Treg cells) ratio was lowered to 15,because of low isolation yields of mononuclear cells from autologousPBMCs. Antibodies 306, 406, 503 and KW0761 were incubated at a singleconcentration of 3.3 nM in triplicates. To confirm that the isolatedautologous PBMCs were functional, the ADCC was performed in parallel onCCR4+CCRF-CEM target cells (data not shown).

The results shown in FIG. 14 and the summarized calculated EC50 values(see Table 24) clearly demonstrate that the anti-CCR4 antibodies of theinvention were able to induce ADCC in the presence of human PBMCs on allthree target cell lines (Hut78 not shown). The most effective anti-CCR4antibody 503 was determined to have an EC50 of 5.3 pM when tested onCCRF-CEM cells, compared to 315 pM of KW0761. Decreased killing activitywas observed for all anti-CCR4 antibodies as described in Example 1 whentested on L-428 cells in comparison to KW0761. This can be explained bythe fact that this cell line was shown to secrete the CCR4-ligandCCL17/TARC, thereby competing with the antibodies binding to CCR4.

In addition, the anti-CCR4 antibodies also exhibited comparable maximumkilling activities when challenged for ADCC on isolated Treg cells (seeFIG. 15).

Example 10 In Vivo Model of Adult T-Cell Lymphoma Leukemia (ATLL)

The anti-CCR4 antibodies described in Example 1 306, 406 and 503 weretested for their ability to reduce tumor growth in a human T-celllymphoma xenograft model using defucosylated IgG1 molecules. AntibodyKW0761, which is based on an antibody previously demonstrated to have invivo efficacy in ATLL (Niwa R et al, CanRes, Vol 65, 2004), was includedas positive control. All experiments described were performed at EPO(Berlin, Germany).

Tumor pieces (3×3×3 mm in size) of the CCR4+ hematological adult T-celllymphoma cell line CCRF-CEM were subcutaneously transplanted into NMRInu/nu mice (Taconic, Hudson, N.Y., USA). In total, 5 different groupswere treated in parallel with the different samples as follows: GroupA=control group (vehicle formulation buffer as described in Example 2),consisting of 5 animals; Group B=KW0761-IgG, consisting of 5 animals;Group C=306-IgG, consisting of 10 animals; Group D=406-IgG, consistingof 10 animals; Group E=503-IgG, consisting of 10 animals. Antibodysamples were applied intravenously (i.v.) when tumors reached a palpablesize of approximate 100 mm3. The tumor volume was calculated usingfollowing equation:

Tumor volume (mm3)=0.5×(major diameter)×(minor diameter)2.

Mice were treated twice a week over 4 weeks (8 treatments in total) at adosage of 20 mg/kg (500 μg of IgG per mouse assuming an average weightof 25 g/mouse). Animals were monitored over a total of 6 weeks after thebeginning of treatment, measuring the tumor volume and body weight twicea week. Animals were sacrificed when tumor sizes exceeded a size of >1.5cm3. The control group had to be sacrificed after 23 days due to largetumor volumes. Statistical significance of obtained data between theexperimental groups were analyzed using software Prism (GraphPad, SanDiego, Calif., USA). Survival proportions were analyzed using Mantel-Coxtest by comparing treated (antibody) versus untreated (control) group.Tumor doubling times were analyzed via analysis of variance (ANOVA) andunpaired t-test analysis.

The results of the study are presented and summarized in FIGS. 16, 17and 18 Treatment of the mice with the antibodies was well tolerated ascould be assessed by the absence of the body weight losses (FIG. 16).Comparison of the individual tumor volumes from the different groups isshown in FIG. 17. It can be seen that the tumor volume within a groupshows high variation, probably due to the fact the tumor model wasinoculated from tumor pieces, which can result in a fast tumor growthafter a lag-phase. The control group A had to be sacrificed after 23days of tumor implantation, due to large tumor sizes. A clear reductionin tumor volume can be seen for all tested anti-CCR4 antibodies. Thelowest mean tumor volume, measured at day 23, in comparison with thecontrol group (treated vs. control in %; T/C) was determined foranti-CCR4 antibody 503 with a value of 35%, 67% in case of KW0761, 50%for 306 and 62% in case of 406, respectively. Statistical significantdifference in the tumor doubling time was also determined for anti-CCR4antibody 503 in comparison with the control group (6.28±1.03 vs.2.58±0.25 days; P=0.0273, unpaired t test). This difference in tumorvolume and tumor doubling time is reflected in the survival curve ofanimals from the same group E (anti-CCR4 antibody 503) for which amedian survival time of 26 days was determined in comparison to 22 daysfor the control group A.

Thus, all three anti-CCR4 antibodies described in Example 1 weredemonstrated to have in vivo tumor eradication efficacy. Statisticalsignificant data were obtained for antibody 503.

Example 11 Aggregation Measurements on Platelets

CCR4 is known to be expressed on platelets, and its ligands MDC and TARCare known to induce platelet aggregation. This could potentially beproblematic, because IgG molecules have two ligand binding sites, sothere is a possibility that an IgG capable of recognizing CCR4 may beable to cross link platelets if both arms are able to bind to CCR4 ondifferent platelets. This might result in blot clotting in vivo.Therefore, the effect of the anti-CCR4 antibodies as described inExample 1 on platelet aggregation were examined. The antibodies wereincubated with isolated platelets alone or in combination ADP, awell-described inducer of aggregation (Varon and Spectre “Antiplateletagents” Hematology Am Soc Hematol Educ Program. 267-72, 2009).

A total of 50 ml of fresh donor blood was collected into sodium citratecontaining tubes. Platelet rich plasma was obtained by centrifuging atroom temperature (RT) for 15 min at 185 g. The platelet containingplasma was transferred into a fresh tube. To define the baseline,platelet depleted plasma was prepared by centrifugation of 1 ml of theplatelet rich plasma for 5 min at 1200 g at RT. The supernatant wastransferred into a new tube and treated in the following the same way asthe platelet rich sample. Aggregation measurements were performed at 37°C. under stirring using an AggRam—aggregometer (Helena Laboratories,Beaumont, Tex., USA). IgGs (503, 406, 306 and anti-GFP as negativecontrol) were incubated with platelets at a concentration of 10 μg/ml inFACS buffer (PBS, pH 7.4, 0.2% BSA, 0.09% NaN3). ADP served as apositive control for aggregation and was used at a final concentrationof 5 μM. The signal obtained with ADP was set as 100%, the baseline 0was defined by the platelet depleted serum, which was measured inparallel in all experimental settings

Binding of the anti-CCR4 antibodies described in Example 1 to plateletswas observed (data not shown). No induction of aggregation was observedand in addition no inhibition of aggregation in presence of ADP wasobserved (data not shown). From these results it can be concluded thatalthough the anti-CCR4 antibodies described in Example 1 bind toplatelets, they have no effect on platelet aggregation. They do notinduce aggregation, nor do they inhibit e.g. ADP-induced plateletaggregation.

Example 12 Binding to Human Renal Cell Carcinoma

CCR4 is known to be involved and to be expressed by solid tumor celllines of clear cell renal cancer cells (CCRC; see patent applicationWO2009/037454). With the aim of demonstrating specific binding to CCR4present in tissues prepared from patients suffering from renal cellcancer (RCC), immune-histochemistry experiments were performed. Allexperiments described were performed at the Centre for TranslationalOncology Institute of Cancer (Barts and the London school of Medicineand Dentistry, Queen Mary University London, Charterhouse Square).Staining was performed using anti-CCR4 antibody 503 and compared to apositive anti-CCR4 antibody.

The RCC tumor microarrays (TMA) from patients with stage IV diagnosiswere provided by the Barts and the London school of Medicine andDentistry (Queen Mary University London, Charterhouse Square, London,UK) and comprised a set of three different cohorts (TMA1, 2 and 3).Paraffin-embedded sections were cut at a thickness of 4 μm, de-waxed andhydrated through alcohol gradient. Antigen retrieval was performed bymicrowaving to boiling point in antigen unmasking solution for 9 minutes(Vectorlaboratories, Burlingame, Calif., USA). Sections were nextblocked in either 5% goat- or 20% rabbit serum (Sigma Aldrich, St Louis,Mo., USA) diluted in PBS for 1 h at RT. For the detection of CCR4, TMAswere stained using the following antibodies: CCR4 was detected usinganti-CCR4 antibody 503 at a concentration of 3 μg/ml and compared togoat anti-CCR4 antibody Ab1669 (3 μg/ml; Abcam, Cambridge, UK). A humanisotype control antibody (Cat. No 1-001-A, used at 3 μg/ml; RnD systems,Minneapolis, Minn. USA) was included to demonstrate binding specificityof anti-CCR4 antibody 503. Antibodies were incubated at 4° C. overnightin corresponding blocking agent (human antibodies in 5% goat serumdiluted in PBS; Ab1669 in 20% rabbit serum in PBS). Slides were washedthree times the following day with PBS in a volume of 30 to 50 ml perwash. Human antibodies 503 and isotype control antibody from RnD weredetected using biotin-conjugated goat anti-human secondary antibody(Cat. No. AP112B, Chemicon International, a division of Millipore,Billerica, Mass., USA) at a dilution of 1:500. Anti-CCR4 antibody Ab1669was detected following incubation with biotin-conjugatedrabbit-anti-goat antibody (BA-1000; Vectorlaboratories, Burlingame,Calif., USA) at a dilution of 1:200 for 45 min at RT. Slides were washedtwice using PBS in a volume of 30 to 50 ml per wash. Endogenousperoxidase was blocked by mixing methanol (Sigma Aldrich, St Louis, Mo.,USA) in mixture with 30% H2O2 for 20 min at RT. The slides were washed 3times using PBS with 30 to 50 ml per wash. Antigen was visualized usingthe vectastain elite ABC kit (Vectorlaboratories, Burlingame, Calif.,USA) and developed using 3,3′-diaminobenzidine (DAB; Sigma Aldrich, StLouis, Mo., USA). Similar staining procedures were performed foranti-CCR4 antibodies 306, 406 and 503 on spleen tissues reported to havehigh expression of CCR4 and thus serving as positive control for theanti-CCR4 antibodies (data not shown). In parallel, detection ofCCR4-ligands TARC and MDC were performed. The staining procedure was asoutlined above, however following antibodies were used. TARC wasdetected using rabbit anti-human-TARC antibody (ab9816, Abcam,Cambridge, UK). The antibody was used at a dilution of 1:50 and detectedusing biotin-conjugated goat anti-rabbit antibody at a dilution of0.1:200 (Vectorlaboratories, Burlingame, Calif., USA). MDC was detectedusing rabbit anti-human-MDC antibody (500-P107, Peprotech, Princeton,N.J., USA) at a dilution of 1:20 and detected using biotin conjugatedanti-rabbitantibody. All binding patterns were analyzed using a NikonEclipse 80i microscope (Nikon, Tokyo, Japan).

Additionally, binding to negative control tissues was assessed for alltested antibodies described above on normal human placenta and kidneytissue.

The results of the staining of tumor microarrays from patients sufferingfrom RCC demonstrate that anti-CCR4 antibody 503 binds to CCR4,expressed in RCC patient tissues in IHC (FIG. 19). Moreover, anti-CCR4antibody 503 was shown to bind to areas of clear cell renal cellcarcinoma, but not papillary renal cell carcinoma (data not shown).Convincingly, the binding pattern overlaps with the anti-CCR4 positivecontrol antibody Ab1669. A slight binding to normal human tissue fromplacenta was observed. However, as the placenta is highly vascularisedorgan, these binding patterns can be referred to blood vessels whereCCR4 is known to be expressed. The expression of CCR4 matched with theexpression pattern of CCR4-ligands TARC and MDC. A summary of thebinding results from the IHC-experiments is presented in Table 25. Thesedata suggest that the anti-CCR4 antibody 503 could serve as therapeuticor diagnostic in RCC.

TABLE 1 SEQ ID NO: scFv 208   7 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSchain FR1:   1 CDR1: SYAMS   8 FR2 WVRQAPGQGLEWMG   2 CDR2:GIIPIFGTVNYAQKFQG   9 FR3: RVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR   3 CDR3:RGGSYFDY  10 FR4: WGQGTLVTVSS  11 Linker: KLSGSASAPKLEEGEFSEARV  12Light SYVLTQPPSASGTPGQSVTISC chainFR1:   4 CDR1: SGSTSNIGSHYVF  13 FR2WYQQLPGTAPRLLIY   5 CDR2: RNHQRPS  14 FR3:GVPDRLSGSKSGTSASLAISGLRSEDEADYYC   6 CDR3: AVWDAKYRGWV  15 FR4:FGGGTKLTVL  56 scFv 208 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGTTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  47 scFv 208QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEW a.a.MGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARRGGSYFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDAKYR GWVFGGGTKLTVL  29V_(H(aa)) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVY YCARRGGSYFDYWGQGTLVTVSS 30 V_(L(aa)) SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDA KYRGWVFGGGTKLTVL 101V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTC (nt)CTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGG GACCCTGGTCACCGTCTCCTCA102 V_(L)domain TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCA (nt)GAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGTTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCT A

TABLE 2 SEQ ID NO: scFv 306   7 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSchain FR1:   1 CDR1: SYAMS   8 FR2 WVRQAPGQGLEWMG   2 CDR2:GIIPIFGTVNYAQKFQG  16 FR3: RVTMTRDTSTSTVYMELSSLRPDDTAVYYCAR   3 CDR3:RGGSYFDY  10 FR4: WGQGTLVTVSS  11 Linker: KLSGSASAPKLEEGEFSEARV  12Light SYVLTQPPSASGTPGQSVTISC chainFR1:  17 CDR1: SGSTSNIGSHYVS  13 FR2WYQQLPGTAPRLLIY   5 CDR2: RNHQRPS  14 FR3:GVPDRLSGSKSGTSASLAISGLRSEDEADYYC   6 CDR3: AVWDAKYRGWV  15 FR4:FGGGTKLTVL  57 scFv 306 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGACCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGTCCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  48 scFv 306QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEW a.a.MGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRPDDTAVYYCARRGGSYFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVSWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDAKYR GWVFGGGTKLTVL  31V_(H(aa)) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRPDDTAVY YCARRGGSYFDYWGQGTLVTVSS 32 V_(L(aa)) SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVSWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDA KYRGWVFGGGTKLTVL 103V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTC (nt)CTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGACCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGG GACCCTGGTCACCGTCTCCTCA104 V_(L)domain TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCA (nt)GAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGTCCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCT A

TABLE 3 SEQ ID NO: scFv 308   7 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSchain FR1:   1 CDR1: SYAMS   8 FR2 WVRQAPGQGLEWMG   2 CDR2:GIIPIFGTVNYAQKFQG  18 FR3: RVTMTRDTSTSTVYMELSSLRPDDTAVYYCAR   3 CDR3:RGGSYFDY  10 FR4: WGQGTLVTVSS  11 Linker: KLSGSASAPKLEEGEFSEARV  12Light SYVLTQPPSASGTPGQSVTISC chainFR1:   4 CDR1: SGSTSNIGSHYVF  13 FR2WYQQLPGTAPRLLIY   5 CDR2: RNHQRPS  14 FR3:GVPDRLSGSKSGTSASLAISGLRSEDEADYYC   6 CDR3: AVWDAKYRGWV  15 FR4:FGGGTKLTVL  58 scFv 308 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGACCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGTTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  49 scFv 308QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEW a.a.MGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRPDDTAVYYCARRGGSYFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDAKYR GWVFGGGTKLTVL  33V_(H(aa)) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRPDDTAVY YCARRGGSYFDYWGQGTLVTVSS 34 V_(L(aa)) SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDA KYRGWVFGGGTKLTVL 105V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTC (nt)CTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGACCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGG GACCCTGGTCACCGTCTCCTCA106 V_(L)domain TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCA (nt)GAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGTTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCT A

TABLE 4 SEQ ID NO: scFv 406  19 Heavy QVQLVQSGAEVKKPGSSVKVSCKASEGTFSchain FR1:   1 CDR1: SYAMS   8 FR2 WVRQAPGQGLEWMG   2 CDR2:GIIPIFGTVNYAQKFQG   9 FR3: RVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR   3 CDR3:RGGSYFDY  10 FR4: WGQGTLVTVSS  11 Linker: KLSGSASAPKLEEGEFSEARV  12Light SYVLTQPPSASGTPGQSVTISC chainFR1:  17 CDR1: SGSTSNIGSHYVS  13 FR2WYQQLPGTAPRLLIY   5 CDR2: RNHQRPS  14 FR3:GVPDRLSGSKSGTSASLAISGLRSEDEADYYC   6 CDR3: AVWDAKYRGWV  15 FR4:FGGGTKLTVL  59 scFv 406 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGAAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGTCCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  50 scFv 406QVQLVQSGAEVKKPGSSVKVSCKASEGTFSSYAMSWVRQAPGQGLEW a.a.MGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARRGGSYFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVSWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDAKYR GWVFGGGTKLTVL  35V_(H(aa)) QVQLVQSGAEVKKPGSSVKVSCKASEGTFSSYAMSWVRQAPGQGLEWMGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVY YCARRGGSYFDYWGQGTLVTVSS 36 V_(L(aa)) SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVSWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDA KYRGWVFGGGTKLTVL 107V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTC (nt)CTCGGTGAAGGTCTCCTGCAAGGCTTCTGAAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGG GACCCTGGTCACCGTCTCCTCA108 V_(L)domain TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCA (nt)GAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGTCCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCT A

TABLE 5 SEQ ID NO: scFv 501  19 Heavy QVQLVQSGAEVKKPGSSVKVSCKASEGTFSchain FR1:   1 CDR1: SYAMS   8 FR2 WVRQAPGQGLEWMG   2 CDR2:GIIPIFGTVNYAQKFQG   9 FR3: RVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR  20 CDR3:RRGAKFDY  10 FR4: WGQGTLVTVSS  11 Linker: KLSGSASAPKLEEGEFSEARV  21Light SYVLTQQPSASGTPGQSVTISC chainFR1:  22 CDR1: SGSTSNIGSHYVV  13 FR2WYQQLPGTAPRLLIY   5 CDR2: RNHQRPS  23 FR3:GVPDRLSGSKSGTSASLAIGGLRSEDEADYYC  24 CDR3: AVWDDTLSGWV  15 FR4:FGGGTKLTVL  60 scFv 501 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGAAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCAACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  51 scFv 501QVQLVQSGAEVKKPGSSVKVSCKASEGTFSSYAMSWVRQAPGQGLEW a.a.MGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARRRGAKFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQQPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLS GWVFGGGTKLTVL  37V_(H(aa)) QVQLVQSGAEVKKPGSSVKVSCKASEGTFSSYAMSWVRQAPGQGLEWMGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVY YCARRRGAKFDYWGQGTLVTVSS 38 V_(L(aa)) SYVLTQQPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDD TLSGWVFGGGTKLTVL 109V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTC (nt)CTCGGTGAAGGTCTCCTGCAAGGCTTCTGAAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGG GACCCTGGTCACCGTCTCCTCA110 V_(L)domain TCCTATGTGCTGACTCAGCAACCCTCAGCGTCTGGGACCCCCGGGCA (nt)GAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCT A

TABLE 6 SEQ ID NO: scFv 503  19 Heavy QVQLVQSGAEVKKPGSSVKVSCKASEGTFSchain FR1:   1 CDR1: SYAMS   8 FR2 WVRQAPGQGLEWMG   2 CDR2:GIIPIFGTVNYAQKFQG   9 FR3: RVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR  20 CDR3:RRGAKFDY  10 FR4: WGQGTLVTVSS  11 Linker: KLSGSASAPKLEEGEFSEARV  12Light SYVLTQPPSASGTPGQSVTISC chainFR1:  22 CDR1: SGSTSNIGSHYVV  13 FR2WYQQLPGTAPRLLIY   5 CDR2: RNHQRPS  23 FR3:GVPDRLSGSKSGTSASLAIGGLRSEDEADYYC  24 CDR3: AVWDDTLSGWV  15 FR4:FGGGTKLTVL  61 scFv 503 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGAAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  52 scFv 503QVQLVQSGAEVKKPGSSVKVSCKASEGTFSSYAMSWVRQAPGQGLEW a.a.MGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARRRGAKFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLS GWVFGGGTKLTVL  39V_(H(aa)) QVQLVQSGAEVKKPGSSVKVSCKASEGTFSSYAMSWVRQAPGQGLEWMGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVY YCARRRGAKFDYWGQGTLVTVSS 40 V_(L(aa)) SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDD TLSGWVFGGGTKLTVL 111V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTC (nt)CTCGGTGAAGGTCTCCTGCAAGGCTTCTGAAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGG GACCCTGGTCACCGTCTCCTCA112 V_(L)domain TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCA (nt)GAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCT A

TABLE 7 SEQ ID NO: scFv 601   7 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSchain FR1:   1 CDR1: SYAMS   8 FR2 WVRQAPGQGLEWMG   2 CDR2:GIIPIFGTVNYAQKFQG   9 FR3: RVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR  20 CDR3:RRGAKFDY  10 FR4: WGQGTLVTVSS  11 Linker: KLSGSASAPKLEEGEFSEARV  21Light SYVLTQQPSASGTPGQSVTISC chainFR1:  22 CDR1: SGSTSNIGSHYVV  13 FR2WYQQLPGTAPRLLIY   5 CDR2: RNHQRPS  23 FR3:GVPDRLSGSKSGTSASLAIGGLRSEDEADYYC  24 CDR3: AVWDDTLSGWV  15 FR4:FGGGTKLTVL  62 scFv 601 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCAACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  53 scFv 601QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEW a.a.MGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARRRGAKFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQQPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLS GWVFGGGTKLTV  41V_(H(aa)) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVY YCARRRGAKFDYWGQGTLVTVSS 42 V_(L(aa)) SYVLTQQPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDD TLSGWVFGGGTKLTV 113V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTC (nt)CTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGG GACCCTGGTCACCGTCTCCTCA114 V_(L)domain TCCTATGTGCTGACTCAGCAACCCTCAGCGTCTGGGACCCCCGGGCA (nt)GAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCT A

TABLE 8 SEQ ID NO: scFv 603   7 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSchain FR1:   1 CDR1: SYAMS   8 FR2 WVRQAPGQGLEWMG   2 CDR2:GIIPIFGTVNYAQKFQG   9 FR3: RVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR  20 CDR3:RRGAKFDY  10 FR4: WGQGTLVTVSS  11 Linker: KLSGSASAPKLEEGEFSEARV  25Light SYVLTQPPSASGTPGQSVTISC chainFR1:  22 CDR1: SGSTSNIGSHYVV  13 FR2WYQQLPGTAPRLLIY   5 CDR2: RNHQRPS  23 FR3:GVPDRLSGSKSGTSASLAIGGLRSEDEADYYC  24 CDR3: AVWDDTLSGWV  15 FR4:FGGGTKLTVL  63 scFv 603 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  54 scFv 603QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEW a.a.MGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARRRGAKFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLS GWVFGGGTKLTVL  43V_(H(aa)) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMGGIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVY YCARRRGAKFDYWGQGTLVTVSS 44 V_(L(aa)) SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDD TLSGWVFGGGTKLTVL 115V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTC (nt)CTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGG GACCCTGGTCACCGTCTCCTCA116 V_(L)domain TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCA (nt)GAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCT A

TABLE 9 SEQ ID NO: scFv 803   7 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSchain FR1:  26 CDR1: SYAIS   8 FR2 WVRQAPGQGLEWMG  27 CDR2:GIIPIFGTANYAQKFQG  28 FR3: RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR  20 CDR3:RRGAKFDY  10 FR4: WGQGTLVTVSS  11 Linker: KLSGSASAPKLEEGEFSEARV  12Light SYVLTQPPSASGTPGQSVTISC chainFR1:  22 CDR1: SGSTSNIGSHYVV  13 FR2WYQQLPGTAPRLLIY   5 CDR2: RNHQRPS  23 FR3:GVPDRLSGSKSGTSASLAIGGLRSEDEADYYC  24 CDR3: AVWDDTLSGWV  15 FR4:FGGGTKLTVL  64 scFv 803 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  55 scFv 803QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEW a.a.MGGIIPIFGTANYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRRGAKFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLS GWVFGGGTKLTVL  45V_(H(aa)) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARRRGAKFDYWGQGTLVTVSS 46 V_(L(aa)) SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDD TLSGWVFGGGTKLTVL 117V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTC (nt)CTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGG GACCCTGGTCACCGTCTCCTCA118 V_(L)domain TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCA (nt)GAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCT A

TABLE 10 SEQ. 208 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT IDIgG1 CGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGC NO: heavyTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGA 65 chainGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGG (nt)GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAASEQ. 208 TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGA ID LambdaGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTA NO: LightTGTGTTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATC 66 chainTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCT (nt)CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCA SEQ. 208QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMG ID IgG-GIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR NO: heavyRGGSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK 67 chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (aa)TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ. 208SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLI ID LambdaYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDAKYRG NO: Light-WVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA 68 chainVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS (aa)CQVTHEGSTVEKTVAPTECS

TABLE 11 SEQ. 306 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT IDIgG1 CGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGC NO: heavyTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGA 69 chainGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGG (nt)GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGACCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAASEQ. 306 TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGA ID LambdaGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTA NO: LightTGTGTCCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATC 70 chainTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCT (nt)CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCA SEQ. 306QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMG ID IgG-GIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRPDDTAVYYCAR NO: heavyRGGSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK 71 chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (aa)TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ. 306SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVSWYQQLPGTAPRLLI ID LambdaYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDAKYRG NO: Light-WVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA 72 chainVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS (aa)CQVTHEGSTVEKTVAPTECS

TABLE 12 SEQ. 308 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT IDIgG1 CGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGC NO: heavyTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGA 73 chainGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGG (nt)GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGACCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAASEQ. 308 TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGA ID LambdaGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTA NO: LightTGTGTTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATC 74 chainTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCT (nt)CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCA SEQ. 308QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMG ID IgG-GIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRPDDTAVYYCAR NO: heavyRGGSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK 75 chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (aa)TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ. 308SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLI ID LambdaYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDAKYRG NO: Light-WVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA 76 chainVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS (aa)CQVTHEGSTVEKTVAPTECS

TABLE 13 SEQ. 406 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT IDIgG1 CGGTGAAGGTCTCCTGCAAGGCTTCTGAAGGCACCTTCAGCAGCTATGC NO: heavyTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGA 77 chainGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGG (nt)GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAASEQ. 406 TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGA ID LambdaGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTA NO: LightTGTGTCCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATC 78 chainTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCT (nt)CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGCGAAATACAGGGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCA SEQ. 406QVQLVQSGAEVKKPGSSVKVSCKASEGTFSSYAMSWVRQAPGQGLEWMG ID IgG-GIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR NO: heavyRGGSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK 79 chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (aa)TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ. 406SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVSWYQQLPGTAPRLLI ID LambdaYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDAKYRG NO: Light-WVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA 80 chainVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS (aa)CQVTHEGSTVEKTVAPTECS

TABLE 14 SEQ. 501 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT IDIgG1 CGGTGAAGGTCTCCTGCAAGGCTTCTGAAGGCACCTTCAGCAGCTATGC NO: heavyTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGA 81 chainGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGG (nt)GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAASEQ. 501 TCCTATGTGCTGACTCAGCAACCCTCAGCGTCTGGGACCCCCGGGCAGA ID LambdaGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTA NO: LightTGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATC 82 chainTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCT (nt)CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCA SEQ. 501QVQLVQSGAEVKKPGSSVKVSCKASEGTFSSYAMSWVRQAPGQGLEWMG ID IgG-GIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR NO: heavyRRGAKFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK 83 chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (aa)TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ. 501SYVLTQQPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLI ID LambdaYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLSG NO: Light-WVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA 84 chainVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS (aa)CQVTHEGSTVEKTVAPTECS

TABLE 15 SEQ. 503 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT IDIgG1 CGGTGAAGGTCTCCTGCAAGGCTTCTGAAGGCACCTTCAGCAGCTATGC NO: heavyTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGA 85 chainGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGG (nt)GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAASEQ. 503 TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGA ID LambdaGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTA NO: LightTGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATC 86 chainTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCT (nt)CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCA SEQ. 503QVQLVQSGAEVKKPGSSVKVSCKASEGTFSSYAMSWVRQAPGQGLEWMG ID IgG-GIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR NO: heavyRRGAKFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK 87 chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (aa)TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ. 503SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLI ID LambdaYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLSG NO: Light-WVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA 88 chainVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS (aa)CQVTHEGSTVEKTVAPTECS

TABLE 16 SEQ. 601 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT IDIgG1 CGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGC NO: heavyTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGA 89 chainGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGG (nt)GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAASEQ. 601 TCCTATGTGCTGACTCAGCAACCCTCAGCGTCTGGGACCCCCGGGCAGA ID LambdaGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTA NO: LightTGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATC 90 chainTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCT (nt)CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCA SEQ. 601QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMG ID IgG-GIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR NO: heavyRRGAKFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK 91 chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (aa)TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ. 601SYVLTQQPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLI ID LambdaYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLSG NO: Light-WVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA 92 chainVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS (aa)CQVTHEGSTVEKTVAPTECS

TABLE 17 SEQ. 603 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT IDIgG1 CGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGC NO: heavyTATGAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGA 93 chainGGGATCATCCCTATCTTTGGTACAGTAAACTACGCACAGAAGTTCCAGG (nt)GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGATGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAASEQ. 603 TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGA ID LambdaGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTA NO: LightTGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATC 94 chainTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCT (nt)CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCA SEQ. 603QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMG ID IgG-GIIPIFGTVNYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCAR NO: heavyRRGAKFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK 95 chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (aa)TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ. 603SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLI ID LambdaYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLSG NO: Light-WVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA 96 chainVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS (aa)CQVTHEGSTVEKTVAPTECS

TABLE 18 SEQ. 803 CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT IDIgG1 CGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGC NO: heavyTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGA 97 chainGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGG (nt)GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACGGCGCGGCGCTAAATTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAASEQ. 803 TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGA ID LambdaGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTA NO: LightTGTGGTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATC 98 chainTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCT (nt)CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCA SEQ. 803QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG ID IgG-GIIPIFGTANYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NO: heavyRRGAKFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK 99 chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (aa)TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ. 803SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVVWYQQLPGTAPRLLI ID LambdaYRNHQRPSGVPDRLSGSKSGTSASLAIGGLRSEDEADYYCAVWDDTLSG NO: Light-WVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA 100 chainVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS (aa)CQVTHEGSTVEKTVAPTECS

TABLE 19 Calculated IC50 values, derived from TARC-mediated Ca-Fluxinhibition. 208 306 308 406 501 503 601 603 803 KM3060 var IC50 (nM)0.0019 0.0012 0.003 0.001 0.001 0.0012 0.003 0.002 0.01 ~172.5 R2 0.980.98 0.99 0.96 0.94 0.93 0.97 0.99 0.99 0.43

TABLE 20 Determined IC₅₀ values from ligand-interfering bindingexperiments of anti-CCR4 IgGs using labelled ligands. IC₅₀ (nM) 306 406503 MDC 0.28 0.27 0.11 R² 0.98 0.99 0.97 TARC 0.51 0.94 0.87 R² 0.960.97 0.95

TABLE 21 Overview of antagonistic properties of anti-CCR4 antibodies inligand mediated Ca-Flux experiments. Inhibition of TARC Inhibition ofMDC IC₅₀ (pM) R² Blockage of signaling in % 306 39 ± 0.4 0.95 74% 406 28± 6  0.97 74% 503 13 ± 3.5 0.96 64% KW0761 n.d. n.d. n.d. *n.d. = notdetermined

TABLE 22 Overview of determined IC₅₀ values and remaining migration in %from inhibition of ligand-induced migration (CCL17/TARC and CCL22/MDC)by anti-CCR4 antibodies. Inhibition of CCL17/TARC Inhibition ofCCL22/MDC Sample IC₅₀ (pM) R² Blockage of migration in % 306 158 0.9750% 406 178 0.98 49% 503 39 0.99 39% KW0761 n.d. n.d. n.d. *n.d. = notdetermined

TABLE 23 Overview of determined IC₅₀ values from inhibition ofCCL17/TARC- induced invasion by anti-CCR4 antibody 503 on 786-O cells.CCL17/TARC CCL17/TARC (125 nM) (25 nM) IC₅₀ (μg/ml) 1.33 0.12 R² 0.970.77

TABLE 24 Overview of determined EC₅₀ values from ADCC experiments onhaematological tumor cell lines CCRF-CEM and L-428. CCRF-CEM L-428 EC₅₀Max. Killing EC₅₀ Max. Killing IgG (pM) (%; nM IgG) (pM) (%; nM IgG) 306114 118 (1.4) 708 88 (7) 406 99.9 121 (1.4) 704 78 (7) 503 5.3 108 (1.4)233 93 (7) KW0761 315 69.8 (1.4) 57 84 (7)

TABLE 25 Scoring table from IHC experiments for renal cell cancer tumormicroarrays (TMA) and relevant control tissues (placenta and normalkidney). Score CCR4 - 503-IgG CCR4 - Ab1669-IgG CCL17 CCL22 Placentacontrol tissue 0 32/39  33/40  38/38 36/37  1 7/39 7/40  0/38 1/37 20/39 0/40  0/38 0/37 Kidney control tissue 0 0/2  1/3  2/3 0/2  1 2/2 2/3  1/3 0/2  2 0/2  0/3  0/3 2/2  Renal cell cancer tissue 0 21/17419/175 128/169 37/171 1 83/174 92/175  39/169 99/171 2 70/174 64/175 2/169 35/171

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1. A nucleic acid molecule comprising a nucleotide sequence encoding anantibody which binds to human CC chemokine receptor 4 (CCR4) and whichis capable of inhibiting the binding of macrophage-derived chemokine(MDC) and/or thymus and activation regulated chemokine (TARC) to CCR4,and which comprises at least one heavy chain variable region thatcomprises three CDRs and at least one light chain variable region thatcomprises three CDRs, wherein said heavy chain variable region comprises(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 1, (ii) a variable heavy (VH) CDR2 that has the amino acidsequence of SEQ ID NO: 2, and (iii) a variable heavy (VH) CDR3 that hasthe amino acid sequence of SEQ ID NO: 20; and wherein said light chainvariable region of said antibody comprises: (iv) a variable light (VL)CDR1 that has the amino acid sequence of SEQ ID NO: 22; (v) a VL CDR2that has the amino acid sequence of SEQ ID NO: 5; and (vi) a VL CDR3that has the amino acid sequence of SEQ ID NO:
 24. 2. A nucleic acidmolecule comprising a nucleotide sequence encoding an antibody whichbinds to human CC chemokine receptor 4 (CCR4) and which is capable ofinhibiting the binding of macrophage-derived chemokine (MDC) and/orthymus and activation regulated chemokine (TARC) to CCR4, wherein saidantibody has a VH domain having the sequence of SEQ ID NO: 39 and/or aVL domain having the sequence of SEQ ID NO:
 40. 3. The nucleic acidmolecule of claim 1, wherein said nucleotide sequence has SEQ ID NO: 111and/or
 112. 4. An expression vector comprising the nucleic acid moleculeof claim
 1. 5. An expression vector comprising the nucleic acid moleculeof claim
 2. 6. An expression vector comprising the nucleic acid moleculeof claim
 3. 7. A host cell or virus comprising the nucleic acid moleculeof claim
 1. 8. A host cell or virus comprising the nucleic acid moleculeof claim
 2. 9. A host cell or virus comprising the nucleic acid moleculeof claim
 3. 10. A method of producing an antibody, comprising: (a)culturing a host cell that comprises an expression vector comprising thenucleic acid molecule of claim 1 under conditions effective to expressthe encoded antibody; and (b) obtaining the expressed antibody from saidhost cell.
 11. A method of producing an antibody, comprising: (c)culturing a host cell that comprises an expression vector comprising thenucleic acid molecule of claim 2 under conditions effective to expressthe encoded antibody; and (d) obtaining the expressed antibody from saidhost cell.
 12. A method of producing an antibody, comprising: (e)culturing a host cell that comprises an expression vector comprising thenucleic acid molecule of claim 3 under conditions effective to expressthe encoded antibody; and (f) obtaining the expressed antibody from saidhost cell.